Protein-based purification matrices and methods of using the same

ABSTRACT

Provided herein are protein-based purification matrices and methods of use thereof to purify biologics and/or to remove contaminants from a composition. Methods of bringing two or more biologics in close proximity are also provided. The disclosed compositions and methods allow for faster, more efficient purification of a biologic compared to traditional affinity chromatography.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 17/493,938,filed Oct. 5, 2021, which is a continuation of International Pat.Application No. PCT/US2021/018805, filed Feb. 19, 2021, which claims thebenefit of priority to U.S. Provisional Application No. 62/978,615,filed Feb. 19, 2020, the contents of which are incorporated by referenceherein in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(ISOL_002_03US_SeqList_ST26.xml; Size: 2,479,551 bytes; and Date ofCreation: Jan. 23, 2023) are herein incorporated by reference in itsentirety.

FIELD

The present disclosure is generally related to compositions and methodsfor purification of biologics. More specifically, the present disclosurerelates to protein-based purification matrices, and methods of using thesame.

BACKGROUND

The use of biologics in medicine and other disciplines is rapidlyincreasing. Biologics often have high affinity and specificity for agiven target, as well as low toxicity and biodegradability. However,their manufacturing and purification can be quite difficult. Biologics,including therapeutic enzymes, antibodies, gene delivery vectors, andother fusion proteins, are typically manufactured recombinantly inbacteria, yeast, or mammalian host cells. Numerous downstreampurification steps are then required in order to meet acceptablestandards of purity (e.g., standards set by the FDA or other regulatorybodies). Host cell proteins, nucleic acids, endotoxins, and viruses areoften the main contaminants that must be removed from biologicpreparations.

One commonly used method of purification is affinity chromatography. Forexample, during antibody manufacture, affinity capture with Protein Achromatography is often the first step after clarification of the cellculture harvest. While affinity chromatography achieves high levels ofpurity (>90%) due to its selectivity for the target biologic, it isexpensive, time consuming, and requires technical equipment that isexpensive to maintain and requires skilled labor. It is also difficultto scale because conditions are not linear as column diameters becomelarge.

There is a need in the art for improved compositions and methods forrapidly and cost-effectively purifying biologics.

SUMMARY

The present disclosure provides protein-based purification matrices forpurifying biologics, and methods for using the same.

In some embodiments, the disclosure provides a method for purifying abiologic, the method comprising contacting the biologic with aprotein-based purification matrix; wherein the biologic binds to thepurification matrix to form a complex; wherein the size of the complexis increased by a first environmental factor; wherein the complex isseparated from at least one contaminant on the basis of size; andwherein the biologic is separated from the purification matrix by asecond environmental factor.

In some embodiments, the disclosure provides a method for removing acontaminant from a composition comprising a biologic, the methodcomprising contacting the contaminant with a protein-based purificationmatrix; wherein the contaminant binds to the matrix to form a complex;wherein the size of the complex is increased by a first environmentalfactor; wherein the complex is separated from the biologic on the basisof size; and wherein the contaminant is separated from the matrix by asecond environmental factor.

In some embodiments, the disclosure provides a method for purifying abiologic, the method comprising contacting the biologic with aprotein-based purification matrix; wherein the biologic binds to thematrix to form a complex; wherein the size of the complex is increased;wherein the complex is separated from at least one contaminant on thebasis of size; and wherein the biologic is separated from the matrix byan environmental factor.

In some embodiments, the disclosure provides a method for separating afirst biologic from a second biologic, the method comprising contactingthe first biologic with a first protein-based purification matrix andcontacting the second biologic with a second protein-based purificationmatrix; wherein the first biologic binds to the first purificationmatrix to form a first complex; wherein the second biologic binds to thesecond purification matrix to form a second complex; and separating thefirst biologic from the second biologic by applying an environmentalfactor.

In some embodiments, the disclosure provides a method of bringing afirst biologic into proximity with a second biologic, the methodcomprising contacting the first biologic with a first protein-basedpurification matrix and contacting the second biologic with a secondprotein-based purification matrix; wherein the first biologic binds tothe first purification matrix to form a first complex; wherein thesecond biologic binds to the second purification matrix to form a secondcomplex; and wherein an environmental factor brings the first complexand the second complex into proximity with one another.

In some embodiments, the purification matrices described herein comprise(i) a capture domain which binds to the biologic, and (ii) a polypeptidewith phase behavior, wherein the capture domain is coupled to thepolypeptide with phase behavior. In some embodiments, the purificationmatrices comprise (i) a capture domain which binds to the contaminant,and (ii) a polypeptide with phase behavior, wherein the capture domainis coupled to the polypeptide with phase behavior.

In some embodiments, the capture domain is coupled to the polypeptidewith phase behavior via a linker. In some embodiments, the linker is apeptide linker. In some embodiments, the peptide linker comprises aprotease cleavage site. In some embodiments, the linker is a chemicallinker.

In some embodiments, the purification matrix comprises a fusion proteincomprising (i) a capture domain which binds to the biologic and (ii) apolypeptide with phase behavior. In some embodiments, the purificationmatrix comprises a fusion protein comprising (i) a capture domain whichbinds to the contaminant and (ii) a polypeptide with phase behavior.

In some embodiments, the polypeptide with phase behavior is aresilin-like polypeptide.

In some embodiments, the polypeptide with phase behavior is anelastin-like polypeptide.

In some embodiments, the polypeptide with phase behavior is a polymercontaining a pentapeptide repeat having the sequence(Val-Pro-Gly-Xaa-Gly)_(n) (SEQ ID NO: 10), or a randomized, scrambledanalog thereof; wherein Xaa can be any amino acid except proline. Insome embodiments, n is an integer from 1 to 360, inclusive of endpoints.

In some embodiments, the polypeptide with phase behavior comprises anamino acid sequence selected from: (GRGDSPY)_(n) (SEQ ID NO: 1);(GRGDSPH)_(n) (SEQ ID NO: 2); (GRGDSPV)_(n) (SEQ ID NO: 3);(GRGDSPYG)_(n) (SEQ ID NO: 4); (RPLGYDS)_(n) (SEQ ID NO: 5);(RPAGYDS)_(n) (SEQ ID NO: 6); (GRGDSYP)_(n) (SEQ ID NO: 7);(GRGDSPYQ)_(n) (SEQ ID NO: 8); (GRGNSPYG)_(n) (SEQ ID NO: 9);(GVGVP)_(n) (SEQ ID NO: 11); (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO:12); (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 13);(GVGVPGWGVPGVGVPGWGVPGVGVP)_(m) (SEQ ID NO: 14);(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m (SEQ ID NO: 15);(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)_(m) (SEQ ID NO: 16); and(GAGVPGVGVPGAGVPGVGVPGAGVP)_(m) (SEQ ID NO: 17); or a randomized,scrambled analog thereof; wherein: n is 20-360; and m is 4-25. In someembodiments, the he polypeptide with phase behavior comprises an aminoacid sequence selected from: (GVGVP)_(m) (SEQ ID NO: 52); (ZZPXXXXGZ)m(SEQ ID NO: 57); (ZZPXGZ)m (SEQ ID NO: 58); (ZZPXXGZ)_(m) (SEQ ID NO:59); or (ZZPXXXGZ)_(m) (SEQ ID NO: 60); wherein m is an integer between10 and 160, inclusive of endpoints, wherein X if present is any aminoacid except proline or glycine, and wherein Z if present is any aminoacid. In some embodiments, the polypeptide with phase behavior comprisesan amino acid sequence of (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO:53) or (GVGVPGVGVPGLGVPGVGVPGVGVP)_(m) (SEQ ID NO: 55); wherein m is aninteger between 2 and 32, inclusive of endpoints. In some embodiments,the polypeptide with phase behavior comprises an amino acid sequenceselected from: (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 193), whereinm is 8 or 16; (GVGVPGAGVP)_(m)(SEQ ID NO: 54), wherein m is an integerbetween 5 and 80, inclusive of endpoints; or (GXGVP)_(m) (SEQ ID NO:56), wherein m is an integer between 10 and 160, inclusive of endpoints,and wherein X for each repeat is independently selected from the groupconsisting of glycine, alanine, valine, isoleucine, leucine,phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid,glutamic acid, and serine.

In some embodiments, the capture domain comprises the sequence of anyone of SEQ ID NO: 24-49, 62-148, and 167-171, or a sequence having atleast 1, at least 2, at least 3, at least 4, or at least 5 mutationsrelative thereto.

In some embodiments, the binding of a biologic to the purificationmatrix is reversible. In some embodiments, the binding of a biologic tothe purification matrix is non-covalent. In some embodiments, thebinding of a biologic to the purification matrix is covalent.

In some embodiments, the binding of a contaminant to the purificationmatrix is reversible. In some embodiments, the binding of a contaminantto the purification matrix is non-covalent. In some embodiments, thebinding of a contaminant to the purification matrix is covalent.

In some embodiments, the biologic is a lipid, a lipopolysaccharide, acell, a protein, a nucleic acid, a carbohydrate, or a virus.

In some embodiments, the biologic is a cell. In some embodiments, thecell is a bacterial cell, a yeast cell, or a mammalian cell. In someembodiments, the cell is a stem cell, a bone cell, a blood cell, amuscle cell, a fat cell, a skin cell, a nerve cell, an endothelial cell,a sex cell, a pancreatic cell, or a cancer cell. In some embodiments,the cell is an immune cell. In some embodiments, the immune cell is a Tcell, a B cell, a NK cell, a peripheral blood mononuclear cell, or aneutrophil. In some embodiments, the cell is a T cell expressing achimeric antigen receptor (CAR).

In some embodiments, the nucleic acid is a DNA or an RNA.

In some embodiments, the virus is an adenovirus, an adeno-associatedvirus (AAV), a lentivirus, a retrovirus, a poxvirus or a herpesvirus.

In some embodiments, the biologic has a diameter between 1 nm and 100µm, inclusive of the endpoints. In some embodiments, the biologic has adiameter between 1 nm and 100 nm, inclusive of the endpoints. In someembodiments, the biologic has a diameter between 100 nm and 1 µm,inclusive of the endpoints. In some embodiments, the biologic has adiameter between 1 µm and 50 µm, inclusive of the endpoints. In someembodiments, the biologic has a diameter between 50 µm and 100 µm,inclusive of the endpoints.

In some embodiments, the methods of the disclosure are completed inabout 0.5 hours to about 24 hours. In some embodiments, the methods ofthe disclosure are completed in about 0.5 hours to about 8 hours. Insome embodiments, the methods of the disclosure are completed in about 2hours to about 6 hours.

In some embodiments, the separation of the complex from the at least onecontaminant can be observed visually with an unaided eye.

In some embodiments, the increase in size of the complex is at least a2-fold increase. In some embodiments, the increase in size of thecomplex is at least a 10-fold increase. In some embodiments, theincrease in size of the complex is at least a 25-fold increase. In someembodiments, the increase in size is an increase in the mass of thecomplex. In some embodiments, the increase in size is an increase in thediameter of the complex.

In some embodiments, the environmental factor comprises one or more of:a change in one or more of temperature, pH, salt concentration,concentration of the purification matrix, concentration of the biologic,or pressure; the addition of one or more surfactants, molecular crowdingagents, reducing agents, oxidizing agents, enzymes, or denaturingagents; or the application of electromagnetic or acoustic waves. In someembodiments, the environmental factor is the first and/or secondenvironmental factor.

In some embodiments, the separation on the basis of size is performedusing tangential flow filtration, membrane chromatography, analyticalultracentrifugation, high performance liquid chromatography, membranechromatography, and/or fast protein liquid chromatography.

In some embodiments, the contaminant is selected from a solvent, anendotoxin, a protein, a peptide, a nucleic acid, and a carbohydrate.

In some embodiments, the purification yield of the biologic is at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%. In some embodiments, the biologic ispurified to at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% purity.

In some embodiments, provided herein is a method of increasing yield ofa biologic during production thereof, the method comprising culturingbiologic-producing cells in the presence of a purification matrix. Insome embodiments, the biologic reversibly binds to the purificationmatrix to form a complex. In some embodiments, the purification matrixis present at a concentration of at least about 10 µM.

In some embodiments, provided herein is a method of stabilizing abiologic during production thereof, the method comprising culturingbiologic-producing cells in the presence of a purification matrix. Insome embodiments, the biologic reversibly binds to the purificationmatrix to form a complex. In some embodiments, the purification matrixis present at a concentration of at least about 10 µM.

In some embodiments, provided herein is a method of stabilizing abiologic during purification thereof, the method comprising contactingthe biologic with a purification matrix during purification thereof. Insome embodiments, the biologic reversibly binds to the purificationmatrix to form a complex. In some embodiments, the purification matrixis present at a concentration of at least about 10 µM.

In some embodiments, provided herein is a method of stabilizing abiologic during storage thereof, the method comprising storing thebiologic in the presence of a purification matrix.

In some embodiments, provided herein is a method of increasing theshelf-life of a biologic, the method comprising storing the biologic inthe presence of a purification matrix. In some embodiments, the biologicreversibly binds to the purification matrix to form a complex. In someembodiments, the purification matrix is present at a concentration of atleast about 10 µM.

These and other embodiments will be further described below in theDetailed Description, Examples, and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the tangential flow filtration flux andtransmembrane pressure during the separation of an AAV9-purificationmatrix complex from impurities. The purification matrix can efficientlypurify AAV9 particles with tangential flow filtration (TFF) inoncentration-diafiltration-concentration-diafiltration (CDCD) mode. Theprocess is performed at high flux and with a low and stabletransmembrane pressure (TMP) in permeate-control mode.

FIG. 2 is a western blot, which shows the presence or absence of VP1,VP2, and VP3 proteins of AAV, at different steps of the tangential flowfiltration (TFF) process. The first lane, labeled SM (starting material)shows the presence of AAV particles in the starting material. The SM issupernatant from cultured HEK293 cells producing a recombinant AAV9vector packaging a tdTomato transgene that is treated with 10 U/mLbenzonase and 0.01 % pluronic acid. The second lane, labeled C (P),shows the absence of AAV particles in the permeate. The third lane,labeled C (R), shows the presence of AAV particles in the retentate. Thefourth lane, labeled W(P), shows the absence of AAV particles in thewash containing removed contaminants. The fifth lane, labeled E(P),shows the eluted AAV particles.

FIG. 3 is a graph showing quantitation of contaminant host cell proteins(HCP) within a composition containing an AAV-purification matrix complexduring TFF concentration and diafiltration. The composition containingthe AAV-purification matrix complex is referred to as SM or “startingmaterial” and contains AAV9 particles. Throughout the concentration anddiafiltration stages of TFF, 1 mL fractions are collected. DV1, DV2,DV3-4, DV5-6, DV7-8, and DV9-10 refer to fractions collected during theconcentration stage of TFF. W1-2, W3-4, and W5-6 refer to fractionscollected during the washing stage of diafiltration. EC, E1, E2, E3, andE4 refer to fractions collected during the elution stage ofdiafiltration.

FIG. 4 is a graph showing the concentration of double stranded DNA(dsDNA) impurities in a composition comprising AAV9 particles beforepurification with a purification matrix (referred to as startingmaterial (SM)) and after purification with a purification matrix(referred to as “elution”).

FIG. 5 is a graph that shows a purification matrix’s efficiency incapturing AAV particles (AAV Capture Efficiency) after clarificationand/or treatment with nuclease. The compositions comprising AAV8particles included compositions from the cell lysate of HEK293 cellsproducing AAV8 particles, referred to as “lysate,” and compositionscomprising media harvested from HEK293 cells producing AAV8 particles,referred to as “supernatant”. The compositions contained viral titers of1 × 10⁷ viral particles per microliter (vp/uL) (referred to as “E7”), 1× 10⁸ vp/uL (“E8”), or 1 × 10¹⁰ vp/uL (“E10”). The compositions wereclarified (+) or not clarified (-). The compositions were exposed tonuclease (+) or not exposed to nuclease (-). A purification matrix wasused to capture AAV8 particles from each composition. The AAV CaptureEfficiency for each sample was calculated using the following equation:100 x (# of AAV8 particles captured by the purification matrix / # ofAAV8 particles in the composition before purification).

FIG. 6 provides an image of a silver stained SDS-PAGE gel, which showsthat purification of AAV8 particles with purification matrix yieldshighly pure AAV8 particles, regardless of pre-treatment with benzonasenuclease. (-) indicates no pre-treatment with benzonase nuclease wasperformed, and (+) indicates that pre-treatment was performed. The firstlane shows molecular weight markers (kDa).

FIG. 7 is a graph that shows dsDNA concentration in AAV8 samples with(+) or without (-) benzonase nuclease pre-treatment. With or withoutpre-treatment, the compositions comprising purified AAV8 eluted from thepurification matrix had similar levels of dsDNA, as assessed by Quant-iTpicogreen assay. Lysed SM: starting material comprised of clarified celllysate; Capture: purification matrix capture step supernatant; Elution:purification matrix elution step supernatant.

FIG. 8 shows AAV Capture Efficiency after centrifugation at variousspeeds. The figure shows that >95% capture of AAV8 particles is achievedusing centrifugation speeds at or greater than 500 relative centrifugalforce (RCF), including 3500 and 16000 RCF.

FIG. 9 shows a comparison of the AAV2 titers produced by HEK293 cellcultures under standard conditions (control) or with the addition ofpurification matrix as quantified by inverted terminal repeat (ITR)quantitative polymerase chain reaction (qPCR) The data shows that thepresence of purification matrix may increase titers by 8% or more.Student’s t-test, p = 0.077.

FIG. 10 shows the percent change of total AAV8 capsids after repeatedfreeze-thaw cycles in a composition comprising AAV8 particles andpurification matrix and a composition comprising AAV8 particles and PBS(negative control).

FIG. 11 is a graph showing the effect of purification matrixconcentration on the Capture Efficiency of AAV8 particles. The CaptureEfficiency was calculated using the following equation: 100 x (# of AAV8particles captured by the purification matrix / # of AAV8 particles inthe composition before purification).

FIG. 12 is a graph showing the effect of purification matrixconcentration on the Capture Efficiency of Ad5 particles. The CaptureEfficiency was calculated using the following equation: 100 x (# of Ad5particles captured by the purification matrix / # of Ad5 particles inthe composition before purification).

FIG. 13 shows fluorescent images of HEK293 cells incubated with thesupernatant of a composition comprising lentivirus particles andphosphate-buffered saline (PBS), labeled LV-GFP control (left) or HEK293cells incubated with the supernatant of a composition comprisinglentivirus particles and purification matrix, labeled LV-GFP withPurification Matrix (right).

FIG. 14 shows an image of an SDS-PAGE gel. The first lane, labeled “0.2um (F)”, contains the filtrate of a complex between human serum albumin(HSA) and purification matrix after introduction of an environmentalfactor (0.6 M NaCl) that is applied to a filter with a 0.2 µm pore size.The second lane, labeled “300 kDa (F)”, contains the filtrate of acomplex between human serum albumin (HSA) and purification matrix afterintroduction of an environmental factor (0.6 M NaCl) that is applied toa filter with a 300 kDa molecular weight cutoff.

FIG. 15 is a graph showing the ability of Ad5 particles to infect HEK293cells (“infectivity”) 0, 3, 7, 11 days after incubation withpurification matrix, matrix control, and PBS (labeled PBS control).

FIG. 16 is a graph showing the ability of Ad5 particles to infect HEK293cells (“infectivity”) incubated with purification matrix or PBS (labeledcontrol) at room temperature (about 25° C.) or 35° C.

FIG. 17 is a graph showing the ability of Ad5 particles to infect HEK293cells (“infectivity”) incubated with purification matrix, matrixcontrol, or PBS (labeled control) after three freeze-thaw cycles (-80°C. to room temperature).

FIG. 18 is an image of SDS-PAGE gels that reveals the presence of IgG1during tangential flow filtration (TFF) concentration (left),diafiltration (left), and elution (right). The composition containingsupernatant of CHO cells expressing IgG1 is referred to as “SM” orstarting material. The SM is concentrated via TFF (“Conc”) and incubatedwith purification matrix “SM + PM” to form a complex. Duringdiafiltration, the complexes are washed with 0.6 M NaCl (labeled“1″-″8”) to remove impurities. The twelfth lane of the SDS-PAGE gel,labeled “wash pool” is a sample from a pool of the wash fractions. IgG1is eluted from the complex with an elution buffer containing 50 mMsodium citrate, 0.6 M NaCl at a pH of 3. The SDS-PAGE gel on the rightshows elution fractions, labeled “1” to “7” and a pool of the elutionfractions labeled “elution pool.”

FIG. 19 shows the flux and transmembrane pressure (TMP) throughouttangential flow filtration (TFF) of a solution containing purificationmatrix and IgG1.

FIG. 20 shows a size exclusion chromatograph of a composition containingimpure IgG1 (top) and of the pure IgG1 composition after purificationvia a purification matrix and tangential flow filtration (bottom).

FIG. 21 shows host cell protein impurities in five differentimmunoglobulin compositions containing IgG2, IgG1, or IgG4 afterpurification using a purification matrix of the disclosure andtangential flow filtration as opposed to purification via protein Achromatography. Host cell protein impurities were quantitated by ELISA.

FIG. 22 shows the productivity of a purification matrix for purifyingimmunoglobulins as described herein, versus two different protein Aresins (MabSelect SuRe™ LX and Amsphere™ A3) (labeled “PrAChromatography Model 1” and “PrA Chromatography Model 2”). Theproductivity is calculated according to the following equation: amountof antibody purified (grams) / unit of material (Liters)/ time (hours).The material refers to the volume of protein A resin or a purificationmatrix of the disclosure.

FIG. 23 shows the percentage of AAV particles captured from cellularsupernatant using a purification matrix, as determined by quantitativepolymerase chain reaction (qPCR).

FIG. 24 shows the percentage of AAV particles eluted from thepurification matrix, as determined by qPCR, using various elutionconditions (e.g., second environmental factors).

FIG. 25 provides an image of a silver-stained gel containing samples of(1) unpurified cellular supernatant, and (2) a sample comprising AAVparticles purified according to the methods of the disclosure. In thesample containing AAV particles, bands for VP1, VP2, and VP3 proteinswere observed at the expected sizes (i.e., 87 kDa, 72 kDa, and 62 kDa,respectively).

FIG. 26 provides an image of a Western Blot, showing that AAV VP1, VP2,and VP3 capsid proteins present in cellular supernatant weresuccessfully captured and removed from the cellular supernatant using apurification matrix described herein.

FIG. 27A shows pictures of cells infected with a control AAV particle(Pos Ctrl), or an AAV8 particle carrying a tdTomato transgene that waseither (i) not purified by the methods of the disclosure (Pos Ctrl) or(ii) purified with a purification matrix of the disclosure (e.g.ViraTag™). Both control AAV and AAV purified with ViraTag™ areinfectious. The image labeled “Neg Ctrl” shows cells which were notinfected with any AAV particles.

FIG. 27B shows the fluorescence intensity of cells infected with acontrol AAV particle (Pos Ctrl) or an AAV particle purified using apurification matrix as described herein (e.g. ViraTag™). Both types ofAAV particles tested carried a tdTomato transgene.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, and in the appended claims, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a protein” canrefer to one protein or to mixtures of such protein, and reference to“the method” includes reference to equivalent steps and/or methods knownto those skilled in the art, and so forth.

As used herein, the term “about” or “approximately” when preceding anumerical value indicates the value plus or minus a range of 10%. Forexample, “about 100” encompasses 90 and 110.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features described herein can be used in any combination.

Moreover, the present disclosure also contemplates that in someembodiments, any feature or combination of features set forth herein canbe excluded or omitted. To illustrate further, if, for example, thespecification indicates that a particular amino acid can be selectedfrom A, G, I, L and/or V, this language also indicates that the aminoacid can be selected from any subset of these amino acid(s) for exampleA, G, I or L; A, G, I or V; A or G; only L; etc., as if each suchsubcombination is expressly set forth herein. Moreover, such languagealso indicates that one or more of the specified amino acids can bedisclaimed. For example, in particular embodiments the amino acid is notA, G or I; is not A; is not G or V; etc., as if each such possibledisclaimer is expressly set forth herein.

As used herein, the term “protein-based” refers to a composition thatcomprises a protein or peptide component. For example, a protein-basedpurification matrix is a purification matrix that comprises a protein orpeptide.

As used herein, the term “environmental factor” is any factor that, whenapplied to a composition comprising a protein-based purification matrix,alters one or more properties of the composition. Non-limiting examplesof environmental factors include a change in one or more of temperature,pH, salt concentration, concentration of the purification matrix,concentration of the biologic, or pressure; the addition of one or moresurfactants, molecular crowding agents, denaturing agents, reducingagents, or oxidizing agents; or the application of electromagnetic oracoustic waves.

As used herein, the term “biologic” may refer to, for example, aprotein, a peptide, a carbohydrate, a nucleic acid, a virus, a cell(e.g., a bacterial, yeast, or mammalian cell), a carbohydrate, a lipid,or a lipopolysaccharide.

As used herein, the term “contaminant” may refer to any substance thatis not desired in a purified composition. In some embodiments, thecontaminant is any substance other than the biologic desired to bepurified. Non-limiting examples of contaminants include, but are notlimited to, a solvent, a protein, a peptide, a carbohydrate, a nucleicacid, a virus, a cell (e.g., a bacterial, yeast, or mammalian cell), acarbohydrate, a lipid, or a lipopolysaccharide. In some embodiments, thecontaminant is an endotoxin or a mycotoxin.

An “adeno-associated virus” (AAV) is a small, replication-deficientparvovirus. As used herein, AAV may refer to a wildtype or mutant AAV ofany one of the following serotypes: AAV1, AAV2, AAV3 (including types 3Aand 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13,AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, avian AAV, bovine AAV,canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV,AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any otherAAV now known or later discovered. In some embodiments, an AAV may havea single-stranded genome, or a double-stranded genome (e.g., aself-complementary AAV). The “capsid” of an AAV particle is anear-spherical protein shell that comprises individual “capsid proteinsubunits” or “capsid proteins” (e.g., about 60 capsid protein subunits)associated and arranged with T=1 icosahedral symmetry. Accordingly, thecapsids of the AAV vectors described herein comprise a plurality ofcapsid proteins. An “AAV particle” typically comprises a capsid, and anucleic acid (e.g., a nucleic acid comprising a transgene) encapsidatedby the protein capsid. When an AAV particle is described as comprising acapsid protein, it will be understood that the AAV particle comprises acapsid, wherein the capsid comprises one or more AAV capsid proteins.When an AAV particle is described as binding to a binding domain, itwill be understood that the binding domain may bind to one or morecapsid proteins within the capsid. The term “empty AAV particle” or“empty capsid” refers to an AAV particle or capsid that does notcomprise any vector genome or nucleic acid comprising an expressioncassette or transgene.

A “viral particle” typically comprises a protein shell (e.g., a capsidor an envelope), and a nucleic acid (e.g., a nucleic acid comprising atransgene) contained therein.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein must contain atleast two amino acids, and no limitation is placed on the maximum numberof amino acids that can comprise a protein’s sequence. The term“peptide” may refer to a short chain of amino acids including, forexample, natural peptides, recombinant peptides, synthetic peptides, ora combination thereof. Proteins and peptides may include, for example,biologically active fragments, substantially homologous polypeptides,oligopeptides, homodimers, heterodimers, variants of polypeptides,modified polypeptides, derivatives, analogs, and fusion proteins, amongothers.

A “polynucleotide” is a sequence of nucleotide bases, and may be RNA,DNA or DNA-RNA hybrid sequences (including both naturally occurringand/or non-naturally occurring nucleotides). In some embodiments, apolynucleotide is either a single or double stranded DNA sequence.

As used herein, by “isolate” or “purify” (or grammatical equivalents) abiologic, it is meant that the biologic is at least partially separatedfrom at least some of the other components in a starting materialcomprising the biologic (e.g., a cell lysate). In representativeembodiments an “isolated” or “purified” biologic is enriched by at leastabout 10-fold, about 100-fold, about 1000-fold, about 10,000-fold ormore as compared with the starting material.

As used herein, the term “polypeptide with phase behavior” refers to anypolypeptide that is capable of undergoing a phase transition. In someembodiments, the polypeptide undergoes a phase transition due to theapplication of an environmental factor. Exemplary polypeptides withphase behavior include elastin-like polypeptides (ELPs) and resilin-likepolypeptides (RLPs).

As used herein, the term “fusion protein” refers to a polypeptideproduced when two heterologous nucleotide sequences or fragments thereofcoding for two (or more) different polypeptides not found fused togetherin nature are fused together in the correct translational reading frame.

As used herein, the term “capture domain” may refer to any amino acidsequence (protein, peptide, etc.) which binds to a target biologic. Insome embodiments, the capture domain may comprise a full-length,truncated, or modified version of a receptor for the target biologic. Insome embodiments, the capture domain may be an antigen-binding portionof a monoclonal antibody (e.g., a Fab), a single-chain variable fragment(scFv) derived from a monoclonal antibody; a natural ligand of thetarget biologic; a peptide with sufficient affinity for the target; asingle domain binder such as a camelid; an artificial binder such as aDarpin; or a single-chain derived from a T-cell receptor.

As used herein, the term “fragment” as it refers to a protein orpolypeptide includes a truncated form of the protein or polypeptide. Forexample, a fragment of CD4 may include about 1 %, about 5 %, about 10 %,about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %,about 45 %, about 50 %, about 55 %, about 60 %, about 65 %, about 70 %,about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 97 %,or about 99 % of the amino acids of full-length CD4.

As used herein, the term “capture efficiency” as it relates to apurification matrix described herein refers to the amount of biologiccaptured by a purification matrix relative to the amount of biologicpresent in the starting composition. The following equation is used todetermine capture efficiency: 100 x (amount of biologic captured by thepurification matrix / amount of biologic in the composition beforepurification).

As used herein, the term “amino acid” encompasses any naturallyoccurring amino acid, modified forms thereof, and synthetic amino acids.Naturally occurring, levorotatory (L-) amino acids are shown in Table 1.

TABLE 1 Amino acid residues and abbreviations. Amino Acid ResidueAbbreviation Three-Letter Code One-Letter Code Alanine Ala A ArginineArg R Asparagine Asn N Aspartic acid (Aspartate) Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid (Glutamate) Glu E Glycine Gly G HistidineHis H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

Alternatively, the amino acid can be a modified amino acid residue(nonlimiting examples are shown in Table 2) and/or can be an amino acidthat is modified by post-translational modification (e.g., acetylation,amidation, formylation, hydroxylation, methylation, phosphorylation orsulfatation).

TABLE 2 Modified Amino Acid Residues Modified Amino Acid ResidueAbbreviation Amino Acid Residue Derivatives 2-Aminoadipic acid Aad3-Aminoadipic acid bAad beta-Alanine, beta-Aminoproprionic acid bAla2-Aminobutyric acid Abu 4-Aminobutyric acid, Piperidinic acid 4Abu6-Aminocaproic acid Acp 2-Aminoheptanoic acid Ahe 2-Aminoisobutyric acidAib 3-Aminoisobutyric acid bAib 2-Aminopimelic acid Apm t-butylalaninet-BuA Citrulline Cit Cyclohexylalanine Cha 2,4-Diaminobutyric acid DbuDesmosine Des 2,21-Diaminopimelic acid Dpm 2,3-Diaminoproprionic acidDpr N-Ethylglycine EtGly N-Ethylasparagine EtAsn Homoarginine hArgHomocysteine hCys Homoserine hSer Hydroxylysine Hyl Allo-HydroxylysineaHyl 3-Hydroxyproline 3Hyp 4-Hydroxyproline 4Hyp Isodesmosine Ideallo-Isoleucine aIle Methionine sulfoxide MSO N-Methylglycine, sarcosineMeGly N-Methyl isoleucine Melle 6-N-Methyllysine MeLys N-MethylvalineMeVal 2-Naphthylalanine 2-Nal Norvaline Nva Norleucine Nle Ornithine Orn4-Chlorophenylalanine Phe(4-C1) 2-Fluorophenylalanine Phe(2-F)3-Fluorophenylalanine Phe(3-F) 4-Fluorophenylalanine Phe(4-F)Phenylglycine Phg Beta-2-thienylalanine Thi

Further, the non-naturally occurring amino acid can be an “unnatural”amino acid (as described by Wang et al., Annu Rev Biophys Biomol Struct.35:225-49 (2006)).

Methods of Using Protein-Based Purification Matrices

The disclosure provides protein-based purification matrices and methodsof using the same. In some embodiments, the protein-based purificationmatrices comprise a capture domain which binds to a target and apolypeptide with phase behavior, wherein the capture domain is coupledto the polypeptide with phase behavior. Non-limiting examples of targetsinclude biologics and contaminants.

In some embodiments, a method of purifying a biologic comprisescontacting the biologic with a protein-based purification matrix;wherein the biologic binds to the purification matrix to form a complex;wherein the size of the complex is increased by a first environmentalfactor; wherein the complex is separated from at least one contaminanton the basis of size; and wherein the biologic is separated from thepurification matrix by a second environmental factor.

In some embodiments, a method of purifying a biologic comprisescontacting the biologic with a protein-based purification matrix;wherein the biologic binds to the matrix to form a complex; wherein thesize of the complex is increased; wherein the complex is separated fromat least one contaminant on the basis of size; and wherein the biologicis separated from the matrix by an environmental factor.

In some embodiments, a method of removing a contaminant from acomposition comprising a biologic comprises contacting the contaminantwith a protein-based purification matrix; wherein the contaminant bindsto the matrix to form a complex; wherein the size of the complex isincreased by a first environmental factor; wherein the complex isseparated from the biologic on the basis of size; and wherein thecontaminant is separated from the matrix by a second environmentalfactor.

In some embodiments, a method of separating a first biologic from asecond biologic comprises contacting the first biologic with a firstprotein-based purification matrix and contacting the second biologicwith a second protein-based purification matrix; wherein the firstbiologic binds to the first purification matrix to form a first complex;wherein the second biologic binds to the second purification matrix toform a second complex; and separating the first biologic from the secondbiologic by applying an environmental factor.

Also provided herein is a method of bringing a biologic in proximity toanother biologic or small molecule. In some embodiments, a method ofbringing a first biologic into proximity with a second biologiccomprises contacting the first biologic with a first protein-basedpurification matrix and contacting the second biologic with a secondprotein-based purification matrix; wherein the first biologic binds tothe first purification matrix to form a first complex; wherein thesecond biologic binds to the second purification matrix to form a secondcomplex; and wherein an environmental factor brings the first complexand second complex into proximity with one another. In some embodiments,a method of bringing a first biologic into proximity with anothermolecule comprises contacting the first biologic with a firstprotein-based purification matrix and contacting the molecule with asecond protein-based purification matrix; wherein the first biologicbinds to the first purification matrix to form a first complex; whereinthe molecule binds to the second purification matrix to form a secondcomplex; and wherein an environmental factor brings the first complexand second complex into proximity with one another. In some embodiments,the methods described herein bring a first biologic and a secondbiologic within about 10 µm, about 5 µm, about 1 µm, about 900 nm, about800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, about300 nm, about 200 nm, about 100 nm, about 10 nm, about 1 nm, about 0.5nm, or about 0.1 nm of one another. In some embodiments, a firstbiologic and a second biologic are brought into proximity with oneanother, wherein the first biologic is an enzyme and the second biologicis a substrate thereof.

In some embodiments, the methods described herein utilize aprotein-based purification matrix comprising a capture domain and apolypeptide with phase behavior. In some embodiments, the methodsdescribed herein utilize two or more distinct protein-based purificationmatrices.

In some embodiments, the methods described herein involve the formationof a complex. In some embodiments, the methods described herein involvethe formation of one or more complexes. In some embodiments, the methodsdescribed herein involve the formation of one, two, three, four, five,or more complexes. The complexes may be referred to as “first complex”or “second complex,” and so on.

In some embodiments, a complex comprises a protein-based purificationmatrix and a biologic. In some embodiments, a complex comprises aprotein-based purification matrix and a contaminant. In someembodiments, a complex comprises a protein-based purification matrix anda second protein such as an enzyme substrate, a metabolite, a ligand(e.g., a ligand that binds to a cellular receptor).

In some embodiments, the components of the complex (e.g. thepurification matrix and the contaminant, biologic, and/or othermolecule) bind to each other. In some embodiments, the binding isreversible. Reversible binding means that the complexes can dissociatee.g. separate into individual components. For example, if a complexreversibly forms between the purification matrix and a biologic, thepurification matrix and the biologic can subsequently disassociate. Insome embodiments, dissociation is triggered by an environmental factor.In some embodiments, reversible binding allows for separation of abiologic from the purification matrix. In some embodiments, reversiblebinding allows for separation of a contaminant from the purificationmatrix. In some embodiments, reversible binding allows for separation ofthe other molecule from the purification matrix.

In some embodiments, reversible binding is non-covalent, i.e. nocovalent bonds are formed between the interacting components of thecomplex (such as between the purification matrix and the contaminant,biologic, and/or other molecule). In some embodiments, non-covalentinteractions cause the purification matrix and the contaminant,biologic, and/or other molecule to bind to each other. Non-limitingexamples of non-covalent interactions include dipole-dipole forces, vander Waals forces, London Dispersion forces, hydrogen bonding,hydrophobic interactions, and electrostatic interactions. In someembodiments, non-covalent binding is disrupted by the addition of anenvironmental factor.

In some embodiments, binding between the purification matrix and atarget molecule (e.g., a contaminant, biologic, and/or other molecule)is covalent. In some embodiments, a covalent bond between a purificationand a matrix may be cleaved using, for example, a protease.

In some embodiments, the size of the complexes described herein increaseafter an environmental factor is applied. In some embodiments, the sizeof a complex formed between the purification matrix and biologic,contaminant, and/or other molecule increases. In some embodiments, thesize of the initial complex increases as a result of aggregation ofmultiple complexes. In some embodiments, multiple complexes aggregatedue to self-assembly of protein-based purification matrices. In someembodiments, multiple complexes aggregate due to the application of anenvironmental factor. In some embodiments, the size increase isstabilized by non-covalent interactions between multiple protein-basedpurification matrix molecules. In some embodiments, the size increase isstabilized by non-covalent interactions between the polypeptides withphase behavior. In some embodiments, the non-covalent interactions aredipole-dipole forces, van der Waals forces, London Dispersion forces,hydrogen bonding, hydrophobic interactions, and/or electrostaticinteractions.

In some embodiments, the methods of the disclosure provide for theformation of multiple complexes in a mixture. In some embodiments, thesize of all of complexes increase. In some embodiments, the size of somecomplexes increases, and the size of the other complexes remainsconstant. In some embodiments, the size of one complex increases, andthe size of the other complex remains constant.

In some embodiments, the size of the initial complex increases by atleast about 2-fold, at least about 5-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 25-fold, atleast about 30-fold, at least about 35-fold, at least about 40-fold, atleast about 45-fold, at least about 50-fold, at least about 55-fold, atleast about 60-fold, at least about 65-fold, at least about 70-fold, atleast about 75-fold, at least about 80-fold, at least about 85-fold, atleast about 90-fold, at least about 95-fold, at least about 100-fold, ormore. In some embodiments, the size of the initial complex increases byat least about 2-fold. In some embodiments, the size of the initialcomplex increases by at least about 5-fold. In some embodiments, thesize of the initial complex increases by at least about 10-fold. In someembodiments, the size of the initial complex increases by at least about25-fold.

As used herein, the phrase “increase in size” may refer to an increasein the diameter of the complex or an increase in the mass of thecomplex. In some embodiments, the increase in size is an increase in themolar mass of the complex. In some embodiments, the increase in size isan increase in the hydrodynamic radius of the complex.

In some embodiments, the increase in size of the complex can be observedvisually with an unaided eye. For example, the increase in size of thecomplex may cause a composition comprising the complex to change color,clarity, viscosity, and/or may cause the complex to change solubility(e.g., to precipitate from solution), wherein such change is observableby a human without the use of any special equipment.

In some embodiments, a person of skill in the art may measure theincrease in the size of the complex according to known methods in theart. In some embodiments, the increase in the size of the complex can bemeasured utilizing a technique selected from the group consisting ofx-ray scattering, small angle x-ray scattering, wide angle x-rayscattering, dynamic light scattering, analytical ultracentrifugation,size exclusion chromatography, and photon correlation spectroscopy.

In some embodiments, the complex of increased size is separated from abiologic, contaminant, and/or small molecule. In some embodiments, thecomplex of increased size containing a biologic and a protein-basedpurification matrix is separated from a contaminant. In someembodiments, the complex of increased size containing a contaminant anda protein-based purification matrix is separated from a compositioncontaining a biologic. In some embodiments, the first complex ofincreased size containing a first biologic and a first protein-basedpurification matrix is separated from a second complex containing asecond biologic and a second protein-based purification matrix.

In some embodiments, separation of the complex from the biologic,contaminant, and/or small molecule can be observed visually with anunaided eye.

In some embodiments, separation of the complex from the biologic,contaminant, and/or small molecule is on the basis of size. In someembodiments, the separation on the basis of size is performed using atechnique selected from the group consisting of tangential flowfiltration (TFF), analytical ultracentrifugation, membranechromatography, high performance liquid chromatography, size exclusionchromatography, membrane chromatography, normal flow filtration,acoustic wave separation, centrifugation, counterflow centrifugation,and fast protein liquid chromatography. In some embodiments, the complexis separated from at least one impurity on the basis of size usingtangential flow filtration. In some embodiments, the complex isseparated from at least one impurity on the basis of size usingcentrifugation. In some embodiments, between about 100 relativecentrifugal force (RCF) and about 16,000 RCF, for example, about 500 toabout 16,000 RCF, about 1,000 RCF to 16,000 RCF, are applied to separatethe complex from at least one impurity. In some embodiments, at least500 relative centrifugal force (RCF) are applied to separate the complexfrom at least one impurity, for example, at least about 500 RCF, atleast about 600 RCF, at least about 700 RCF, at least about 800 RCF, atleast about 900 RCF, at least about 1000 RCF, at least about 2000 RCF,at least about 3000 RCF, at least about 3500 RCF, at least about 4000RCF, at least about 5000 RCF, at least about 6000 RCF, at least about7000 RCF, at least about 8000 RCF, at least about 9000 RCF, at leastabout 10,000 RCF, at least about 11,000 RCF, at least about 12,000 RCF,at least about 13,000 RCF, at least about 14,000 RCF, at least about15,000 RCF, at least about 16,000 RCF, at least about 17,000 RCF, atleast about 18,000 RCF, at least about 19,000 RCF, or at least about20,000 RCF.

In some embodiments, separation of the complex from the biologic,contaminant, and/or small molecule on the basis of size is performedusing TFF. In some embodiments, TFF may be used to separate the complexfrom at least one impurity on the basis of size, a process also referredto herein as “diafiltration.” Diafiltration comprises both washing andelution steps. Washing removes impurities contained in the compositioncomprising the complexes. Elution separates purified biologics from thepurification matrix. In some embodiments, the complexes are concentratedusing TFF. In some embodiments, TFF may be used to increase theconcentration of a complex within a composition, a process also referredto herein as “concentration.”

Tangential flow filtration employs both microfiltration andultrafiltration membranes to separate molecules. Microfiltrationmembranes typically have pore sizes between 0.1 µm and 10 µm.Ultrafiltration membranes typically have smaller pore sizes thanmicrofiltration membranes with pore sizes between 0.001 µm and 0.1 µm.In some embodiments, a membrane with a pore size between about 0.001 µmand about 10 µm is utilized in the methods of the disclosure. In someembodiments, the membrane has a pore size of about 0.001 µm, about 0.01µm, about 0.05 µm, about 0.1 µm, about 0.2 µm, about 0.3 µm, about 0.4µm, about 0.5 µm, about 0.6 µm, about 0.7 µm, about 0.8 µm, about 0.9µm, about 1.0 µm, about 2 µm, about 3 µm, about 4 µm, about 5 µm, about6 µm, about 7 µm, about 8 µm, about 9 µm, or about 10 µm, including allvalues and ranges in between thereof. In some embodiments, the membranehas a pore size of about 0.1 µm. In some embodiments, the membrane has apore size of about 0.2 µm.

In some embodiments, the membrane is made of hydrophilizedpoly(vinylildene difluoride) (PVDF), polyetheresulfone (PES), cellulosephosphate, diethylaminoethyl cellulose, polysufone, regeneratedcellulose, nylon, cellulose nitrate, cellulose acetate, pegylated PES,modified polyethersulfone, and sulfonated PES, or modified derivativesthereof of the aforementioned materials.

In TFF, a membrane is placed tangentially to the flow of a fluid mixtureto cause the fluid mixture to flow tangentially over a first side of themembrane. At the same time, a fluid media is placed in contact with asecond surface of the membrane. A transmembrane pressure is the forcethat drives fluid through the membrane, carrying along permeablemolecules.

In some embodiments, separation of the complex from the biologic,contaminant, and/or small molecule on the basis of size is performedusing TFF with a transmembrane pressure of between about 0.1 bar toabout 3 bar. In some embodiments, the transmembrane pressure is about0.1 bar, about 0.2 bar, about 0.3 bar, about 0.4 bar, about 0.5 bar,about 0.6 bar, about 0.7 bar, about 0.8 bar, about 0.9 bar, about 1.0bar, about 1.1 bar, about 1.2 bar, about 1.3 bar, about 1.4 bar, about1.5 bar, about 1.6 bar, about 1.7 bar, about 1.8 bar, about 1.9 bar,about 2.0 bar, about 2.1 bar, about 2.2 bar, about 2.3 bar, about 2.4bar, about 2.5 bar, about 2.6 bar, about 2.7 bar, about 2.8 bar, about2.9 bar, or about 3.0 bar, including all values and ranges in between.In some embodiments, the transmembrane pressure is about 1.5 bar.

In some embodiments, the cross flow rate is tuned to improve theseparation of the complexes described herein from the biologic,contaminant, and/or small molecule. The cross flow rate is the rate ofsolution flow through the feed channel and across the membrane. Itprovides the force that sweeps away molecules that can restrict filtrateflow. In some embodiments, the cross flow rate is between about 500L/m²/h and about 2000 L/m²/h. In some embodiments, the cross flow rateis between about 500 L/m²/h, about 600 L/m²/h, about 700 L/m²/h, about800 L/m²/h, about 900 L/m²/h, about 1000 L/m²/h, about 1100 L/m²/h,about 1200 L/m²/h, about 1300 L/m²/h, about 1400 L/m²/h, about 1500L/m²/h, about 1600 L/m²/h, about 1700 L/m²/h, about 1800 L/m²/h, about1900 L/m²/h, or about 2000 L/m²/h, including all values and ranges inbetween thereof. In some embodiments, the cross flow rate is about 960L/m²/h. In some embodiments, TFF separation occurs by using a membranethat retains the complex containing the purification matrix and thebiologic while passing the contaminant. In some embodiments, a membranethat retains the complex containing the purification matrix and thecontaminant while passing the biologic is used. In some embodiments, amembrane that retains the complex containing the purification matrix andthe contaminant while passing the biologic is utilized. In someembodiments, a membrane that retains the first complex containing thepurification matrix and the first biologic while passing the complexcontaining the second purification matrix and second biologic isutilized.

In some embodiments, the methods described herein enable thepurification of at least 0.1 kg, at least about 0.2 kg, at least about0.3 kg, at least about 0.4 kg, at least about 0.5 kg, at least about 0.6kg, at least about 0.7 kg, at least about 0.8 kg, at least about 0.9 kg,at least about 1 kg, at least about 2 kg, at least about 3 kg, at leastabout 4 kg, at least about 5 kg, at least about 6 kg, at least about 7kg, at least about 8 kg, at least about 9 kg, at least about 10 kg, ormore of biologic per day, including all values and ranges in between.

In some embodiments, the methods described herein are completed in about0.5 hr to about 24 hours. In some embodiments, the methods are completedin about 0.5 hr, about 1 hr, about 2 hr, about 3 hr, about 4 hr, about 5hr, about 6 hr, about 7 hr, about 8 hr, about 9 hr, about 10 hr, about11 hr, about 12 hr, about 13 hr, about 14 hr, about 15 hr, about 16 hr,about 17 hr, about 18 hr, about 19 hr, about 20 hr, about 21 hr, about22 hr, about 23 hr, or about 24 hr. In some embodiments, the methodsdescribed herein are completed in about 0.5 hr to about 8 hr. In someembodiments, the methods of the disclosure are completed in about 2 hrto about 6 hr.

In some embodiments, the purification yield of the biologic is at least70 %, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%.

In some embodiments, the biologic is purified to at least 70%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% purity.

In some embodiments, the purified biologic retains its biologicalactivity and/or structure. In some embodiments, the purified biologichas enhanced biological activity.

In some embodiments, at least 400 g/m² (grams of biologic per m² offilter membrane) are purified per day. In some embodiments, at least 400g/m², at least 500 g/m², at least 600 g/m², at least 700 g/m², at least800 g/m², at least 900 g/m², or at least 1000 g/m² of biologic arepurified per day. In some embodiments, the biologic is a protein. Insome embodiments, the protein is an antibody. In some embodiments, thebiologic is a virus, such as an AAV, adenovirus, or lentivirus.

In some embodiments, at least about 150 g/L (grams of biologic perliters of protein-based purification matrix) are purified per day. Insome embodiments, at least about 150 g/L, at least about 200 g/L, atleast about 250 g/L, at least about 300 g/L, at least about 350 g/L, atleast about 400 g/L, at least about 450 g/L, at least about 500 g/L, atleast about 550 g/L, at least about 600 g/L, at least about 650 g/L, atleast about 700 g/L, at least about 750 g/L, at least about 800 g/L, atleast about 850 g/L, at least about 900 g/L, at least about 950 g/L, orat least about 1000 g/L are purified per day. In some embodiments, thebiologic is a protein. In some embodiments, the protein is an antibody.In some embodiments, the biologic is a viral particle, such as an AAVparticle.

Methods for Stabilizing Biologics

The instant inventors have discovered that the purification matricesdescribed herein may unexpectedly help stabilize biologics duringproduction, purification, and/or storage thereof. As used herein withrelation to a biologic, the terms “stabilize” or “stabilizing” refers tothe ability of a purification matrix to reduce degradation oraggregation of a biologic sample comprising a plurality of biologicmolecules, to prevent biologic molecules from binding other proteins, orto enhance synthesis of a biologic by a producer cell.

Thus, in some embodiments, a biologic is contacted with a purificationmatrix during production, purification, or storage thereof. In someembodiments, a method of increasing yield of a biologic duringproduction thereof comprises culturing biologic-producing cells in thepresence of a purification matrix. In some embodiments, a method ofstabilizing biologic during production thereof comprises culturingbiologic-producing cells in the presence of a purification matrix. Insome embodiments, a method of stabilizing biologics during purificationthereof comprises contacting the biologic with a purification matrixduring purification thereof. In some embodiments, a method ofstabilizing a biologic during storage thereof comprises storing thebiologic in the presence of a purification matrix. In some embodiments,a method of increasing the shelf-life of a biologic comprises storingthe biologic in the presence of a purification matrix.

For example, a purification matrix may be contacted with a biologicduring production of the biologic in culture. Biologics such as viralparticles and proteins (e.g., monoclonal antibodies), for example, maybe produced in production cell lines, such as HEK293 cells. In someembodiments, the production cell lines are transfected with one or moreplasmids containing various genes required to produce a biologic. Addingthe purification matrix to the biologic-producing cells in culture(e.g., by adding it to the tissue culture media) may increase the yieldand/or quality of the biologic obtained during this process. In someembodiments, the purification matrix may be added to the culture at aconcentration of about 1 µM to about 1 mM, for example, about 1 µM,about 2 µM, about 3 µM, about 4 µM, about 5 µM, about 6 µM, about 7 µM,about 8 µM, about 9 µM, about 10 µM, about 20 µM, about 30 µM, about 40µM, about 50 µM, about 60 µM, about 70 µM, about 80 µM, about 90 µM,about 100 µM, about 150 µM, about 200 µM, about 250 µM, about 300 µM,about 350 µM, about 400 µM, about 450 µM, about 500 µM, about 550 µM,about 600 µM, about 650 µM, about 700 µM, about 750 µM, about 800 µM,about 850 µM, about 900 µM, about 950 µM, or about 1 mM, including allvalues and ranges in between. In some embodiments, the purificationmatrix is added to culture at a concentration of about 10 µM. In someembodiments, the purification matrix is added to culture at aconcentration of about 100 µM.Without being bound by any theory, it isbelieved that the purification matrix can bind to and/or physicallysurround the biologic molecules as they are produced, thereby preventingthem from binding other proteins. Thus, a protein or viral particleproduced by a cultured cell and secreted into culture medium, in thepresence of purification matrix, would not be able to re-bind to areceptor on a producer cell. In some embodiments, adding a purificationmatrix to biologic-producer cell lines in culture may increase the yieldof the biologic. For example, adding a purification matrix to aviral-producing cell line in culture, such as a cell line producinglentiviral particles or adenoviral particles, may result in an increasein viral titer obtained by at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least100-fold, or more, compared to cells cultured without the purificationmatrix. Adding a purification matrix to a protein-producing cell line inculture may result in an increase in yield of protein obtained by atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, atleast 10-fold, at least 50-fold, at least 100-fold, or more, compared tocells cultured without the purification matrix.

As another example, after a purified biologic composition is prepared(using the methods described herein, or other methods known in the art),a purification matrix may be added to the sample before storage (e.g.,freezing). Without being bound by any theory, it is believed that thepurification matrix can bind to and/or physically surround biologicmolecules, thus preventing them from aggregating with other biologicmolecules, and also helping to protect them from degradation,particularly during multiple freeze-thaw cycles. Aggregation of biologicmolecules may be observed visually by microscopy and/or by a techniqueselected from the group consisting of x-ray scattering, laserdiffraction, analytical ultracentrifugation, dynamic light scattering,nanoparticle tracking analysis, resonant mass measurement, sizeexclusion chromatography, gel permeation chromatography, lightobscuration, and combinations thereof. In some embodiments, the biologicmay be frozen and stored in the presence of a purification matrix attemperatures between about -80° C. and about 40° C., for example, about-80° C., about -75° C., about -70° C., about -65° C., about -60° C.,about -55° C., about -50° C., about -45° C., about -40° C., about -35°C., about -30° C., about -25° C., about -20° C., about -15° C., about-10° C., about -5° C., about 0° C., about 4° C., about 5° C., about 10°C., about 15° C., about 20° C., about 25° C., about 30° C., about 35°C., or about 40° C.

In some embodiments, when a biologic is stored in the presence of apurification matrix, the shelf life of the biologic is at least about 10% longer as compared to a sample stored in the absence of purificationmatrix. For example, in some embodiments, the shelf life is at leastabout 10 %, at least about 20 %, at least about 30 %, at least about 40%, at least about 50 %, at least about 60 %, at least about 70 %, atleast about 80 %, at least about 90 %, at least about 100 %, at leastabout 150 %, at least about 200 %, at least about 250 %, at least about300 %, at least about 350 %, at least about 400 %, at least about 450 %,or at least about 500 % longer than the shelf life of a biologic storedin the absence of a purification matrix at about the same temperature.

In some embodiments, a biologic is stored at about -80° C. in thepresence of a purification matrix. In some embodiments, a biologic isstored at about -20° C. in the presence of a purification matrix. Insome embodiments, a biologic is stored at about 4° C. in the presence ofa purification matrix. In some embodiments, a biologic is stored atabout 37° C. in the presence of a purification matrix. In someembodiments, when a biologic is stored in the presence of a purificationmatrix at about -80° C., about -20° C., about 4° C., or about 37° C.,the shelf life of the biologic is at least about 10 % longer than if itwas stored in the absence of the purification matrix. For example, theshelf life of the biologic may be at least about 10 %, at least about 20%, at least about 30 %, at least about 40 %, at least about 50 %, atleast about 60 %, at least about 70 %, at least about 80 %, at leastabout 90 %, at least about 100 %, at least about 150 %, at least about200 %, at least about 250 %, at least about 300 %, at least about 350 %,at least about 400 %, at least about 450 %, or at least about 500 %longer than the shelf life of the biologic stored in the absence of apurification matrix at the same temperature. As used herein, increasedshelf life may refer to an increase in the amount of time a biologic isstored and still retains its function. For example, a viral particlethat retains its function shares substantially the same level ofinfectivity.

Protein-Based Purification Matrices Capture Domains

The protein-based purification matrices described herein may comprise acapture domain. In some embodiments, the capture domain binds abiologic. Non-limiting examples of biologics include a cell, a lipid, alipopolysaccharide, a protein, a nucleic acid, a carbohydrate, or avirus.

In some embodiments, the capture domain binds a cell. In someembodiments, the cell is selected from the group consisting of abacterial cell, yeast cell, or an animal cell such as a mammalian cell.In some embodiments, the cell is a chicken cell, a mouse cell, a guineapig cell, a rat cell, a rabbit cell, a goat cell, a horse cell, a sheepcell, a dog cell, a cat cell, or a cow cell. In some embodiments, thecell is a human cell.

In some embodiments, the cell is a stem cell, a bone cell, a blood cell,a muscle cell, a fat cell, a skin cell, a nerve cell, an endothelialcell, a sex cell, a pancreatic cell, or a cancer cell.

In some embodiments, the cell is an immune cell. In some embodiments,the immune cell is a T cell, a B cell, a NK cell, a peripheral bloodmononuclear cell, monocyte, macrophage, or a neutrophil. In someembodiments, the cell is a T cell expressing a chimeric antigen receptor(CAR).

In some embodiments, the capture domain binds a protein. In someembodiments, the protein is fibrous, globular, or a membrane protein. Insome embodiments, the protein is an antibody.

In some embodiments, the capture domain binds an antibody orantigen-binding portion of a monoclonal antibody. In some embodiments,the capture domain binds to a fragment crystalline (Fc) region. In someembodiments, the Fc region is part of an antibody or antigen-bindingportion of a monoclonal antibody. In some embodiments, the capture isProtein A or Protein G, which are known to bind to a Fc region.

In some embodiments, the capture domain comprises protein A, or aderivative or fragment thereof. Protein A (see, e.g., Uniprot AccessionNo. Q70AB8), binds to the Fc region of most immunoglobulins. The aminoacid sequence of Protein A is:

(M)AAQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKEEDNNKPGKEDGNKPGKEDGN (SEQ ID NO: 126).

In some embodiments, the capture domain comprises Protein A having anamino acid sequence of SEQ ID NO: 126 with a mutation of A117G. In someembodiments, the capture domain comprises the B domain of Protein A or afragment or derivative thereof, having the amino acid sequence of SEQ IDNO: 127. In some embodiments, the capture domain comprises the B domainof Protein A or a fragment or derivative thereof, having the amino acidsequence of SEQ ID NO: 127 with an amino acid mutation of A2G. In someembodiments, the capture domain comprises the C domain of Protein A or afragment or derivative thereof, having the amino acid sequence of SEQ IDNO: 128. In some embodiments, the capture domain comprises the Z domainof Protein A or a fragment or derivative thereof, having the amino acidsequence of SEQ ID NO: 179. In some embodiments, the capture domaincomprises the amino acid sequence of SEQ ID NO: 171 or an amino acidsequence having at least 90 %, at least 95 %, at least 96 %, at least 97%, at least 98 %, or at least 99 % identity to SEQ ID NO: 171.

In some embodiments, the capture domain comprising Protein A, or afragment or derivative thereof, comprises the amino acid sequence of SEQID NOs: 126-128 and 171 with at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25 or moreamino acid mutations. In some embodiments, the capture domain comprisingProtein A, or fragment or derivative thereof, comprises a sequence withat least 90%, at least 95%, at least 97 %, or at least 99 % identity toany one of SEQ ID NOs: 126-128 and 171.

Unless otherwise indicated, sequence identity is determined using theNational Center for Biotechnology Information (NCBI)′s Basic LocalAlignment Search Tool (BLAST^(®)), available atblast.ncbi.nlm.nih.gov/Blast.cgi. In some embodiments, the sequenceidentity is calculated over the entire length of the compared sequences.In some embodiments, the sequence identity is calculated over a 20-aminoacid, 50-amino acid, 75-amino acid, 100-amino acid, 250-amino acid,500-amino acid, 750-amino acid, or 1000-amino acid fragment of eachcompared sequence.

In some embodiments, the capture domain comprises protein G, or aderivative or fragment thereof. Protein G (see, e.g., Uniprot AccessionNo. P19909), binds to the Fc region of most immunoglobulins. The aminoacid sequence of Protein G is:

(M)EKEKKVKYFLRKSAFGLASVSAAFLVGSTVFAVDSPIEDTPIIRNGGELTNLLGNSETTLALRNEESATADLTAAAVADTVAAAAAENAGAAAWEAAAAADALAKAKADALKEFNKYGVSDYYKNLINNAKTVEGVKDLQAQVVESAKKARISEATDGLSDFLKSQTPAEDTVKSIELAEAKVLANRELDKYGVSDYHKNLINNAKTVEGVKDLQAQVVESAKKARISEATDGLSDFLKSQTPAEDTVKSIELAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEMVTEVPGDAPTEPEKPEASIPLVPLTPATPIAKDDAKKDDTKKEDAKKPEAKKEDAKKAETLPTTGEGSNPFFTAAALAVMAGAGALAVASKRKED (SEQ  ID NO: 131).

In some embodiments, the capture domain comprises the G domain ofProtein G or a fragment or derivative thereof, having the amino acidsequence of SEQ ID NO: 132. In some embodiments, the capture domaincomprises a fragment of Protein G, having the amino acid sequence of SEQID NO: 133.

In some embodiments, the capture domain comprising Protein G, or afragment or derivative thereof, comprises the amino acid sequence of SEQID NOs: 131-133 with at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25 or more amino acidmutations. In some embodiments, the capture domain comprising Protein G,or fragment or derivative thereof, comprises a sequence with at least90%, at least 95%, at least 97 %, or at least 99 % identity to any oneof SEQ ID NOs: 131-133.

In some embodiments, the capture domain binds to a kappa light chain. Insome embodiments, the kappa light chain is part of an antibody orantigen-binding portion of a monoclonal antibody thereof. In someembodiments, the capture domain comprises protein L. Protein L binds toantibodies or antibody binding fragments thereof through interactionswith the kappa light chain. The amino acid sequence of Protein L is:

(M)KINKKLLMAALAGAIVVGGGANAYAAEEDNTDNNLSMDEISDAYFDYHGDVSDSVDPVEEEIDEALAKALAEAKETAKKHIDSLNHLSETAKKLAKNDIDSATTINAINDIVARADVMERKTAEKEEAEKLAAAKETAKKHIDELKHLADKTKELAKRDIDSATTINAINDIVARADVMERKTAEKEEAEKLAAAKETAKKHIDELKHLADKTKELAKRDIDSATTIDAINDIVARADVMERKLSEKETPEPEEEVTIKANLIFADGSTQNAEFKGTFAKAVSDAYAYADALKKDNGEYTVDVADKGLTLNIKFAGKKEKPEEPKEEVTIKVNLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKENGEYTADLEDGGNTINIKFAGKETPETPEEPKEEVTIKVNLIFADGKIQTAEFKGTFEEATAKAYAYANLLAKENGEYTADLEDGGNTINIKFAGKETPETPEEPKEEVTIKVNLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVNGEYTADLEDGGYTINIKFAGKEQPGENPGITIDEWLLKNAKEEAIKELKEAGITSDLYFSLINKAKTVEGVEALKNEILKAHAGEETPELKDGYATYEEAEAAAKEALKNDDVNNAYEIVQGADGRYYYVLKIEVADEEEPGEDTPEVQEGYATYEEAEAAAKEALKEDKVNNAYEVVQGADGRYYYVLKIEDKEDEQPGEEPGENPGITIDEWLLKNAKEDAIKELKEAGISSDIYFDAINKAKTVEGVEALKNEILKAHAEKPGENPGITIDEWLLKNAKEAAIKELKEAGITAEYLFNLINKAKTVEGVESLKNEILKAHAEKPGENPGITIDEWLLKNAKEDAIKELKEAGITSDIYFDAINKAKTIEGVEALKNEILKAHKKDEEPGKKPGEDKKPEDKKPGEDKKPEDKKPGEDKKPEDKKPGKTDKDSPNKKKKAKLPKAGSEAEILTLAAAALSTAAGAYVSLKKRK (SEQ  ID NO:129).

In some embodiments, the capture domain comprises a fragment of ProteinL having an amino acid sequence of SEQ ID NO: 130.

In some embodiments, the capture domain comprising Protein L, or afragment or derivative thereof, comprises the amino acid sequence of SEQID NOs: 129-130 with at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25 or more amino acidmutations. In some embodiments, the capture domain comprising Protein L,or fragment or derivative thereof, comprises a sequence with at least90%, at least 95%, at least 97 %, or at least 99 % identity to any oneof SEQ ID NOs: 129-130.

In some embodiments, the capture domain binds an albumin, or derivativeor fusion thereof. In some embodiments, the albumin is human serumalbumin (HSA), bovine serum albumin (BSA), or ovalbumin. In someembodiments, the capture domain that binds an albumin comprisesalbumin-binding polypeptide, or a fragment or derivative thereof. Insome embodiments, the albumin-binding polypeptide comprises an aminoacid sequence of SEQ ID NO: 125. In some embodiments, the capture domaincomprising an albumin-binding polypeptide, or fragment or derivativethereof, comprises the amino acid sequence of SEQ ID NO: 125 with atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25 or more amino acid mutations. In someembodiments, the capture domain comprising an albumin bindingpolypeptide, or fragment or derivative thereof, comprises a sequencewith at least 90%, at least 95%, at least 97 %, or at least 99 %identity to SEQ ID NO: 125. In some embodiments, the capture domain thatbinds to albumin has an amino acid sequence of SEQ ID NO: 170, or anamino acid sequence with at least 90 %, at least 95 %, at least 96 %, atleast 97 %, at least 98 %, or at least 99 % identity to SEQ ID NO: 170.

In some embodiments, the capture domain binds a nucleic acid. In someembodiments, the nucleic acid is a DNA or an RNA. In some embodiments,the nucleic acid is a DNA/RNA hybrid.

In some embodiments, the capture domain is a protein that binds to anucleic acid. In some embodiments, the protein that binds to a nucleicacid binds to an adenosine and uridine rich element (ARE) of mRNA, amRNA cap, a poly(A) tail, RNA, or double stranded DNA.

In some embodiments, the capture domain comprises mRNA decay activatorprotein ZFP36L2 (Tis11d), or a fragment or derivative thereof. Tis11d G(see, e.g., Uniprot Accession No. P47974) binds to an ARE. The aminoacid sequence of Tis11d is:(M)STTLLSAFYDVDFLCKTEKSLANLNLNNMLDKKAVGTPVAAAPSSGFAPGFLRRHSASNLHALAHPAPSPGSCSPKFPGAANGSSCGSAAAGGPTSYGTLKEPSGGGGTALLNKENKFRDRSFSENGDRSQHLLHLQQQQKGGGGSQINSTRYKTELCRPFEESGTCKYGEKCQFAHGFHELRSLTRHPKYKTELCRTFHTIGFCPYGPRCHFIHNADERRPAPSGGASGDLRAFGTRDALHLGFPREPRPKLHHSLSFSGFPSGHHQPPGGLESPLLLDSPTSRTPPPPSCSSASSCSSSASSCSSASAASTPSGAPTCCASAAAAAAAALLYGTGGAEDLLAPGAPCAACSSASCANNAFAFGPELSSLITPLAIQTHNFAAVAAAAYYRSQQQQQQQGLAPPAQPPAPPSATLPAGAAAPPSPPFSFQLPRRLSDSPVFDAPPSPPDSLSDRDSYLSGSLSSGSLSGSESPSLDPGRRLPIFSRLSISDD (SEQ ID NO: 134). The methionine enclosed by parenthesisin the aforementioned sequence (M) or in any other sequences describedherein is an initiator methionine. The presence of an initiatormethionine is optional.

In some embodiments, the capture domain comprises a Tis11d fragmenthaving an amino acid sequence of SEQ ID NO: 135. In some embodiments,the capture domain comprises the RNA binding domain of Tis11d having anamino acid sequence of SEQ ID NO: 136.

In some embodiments, the capture domain comprises Tis11d having an aminoacid sequence of SEQ ID NO: 134 with at least one mutation selected fromE195D, E195H, E195G, E195A, E195R, and E195K. In some embodiments, thecapture domain comprises a Tis11d fragment having an amino acid sequenceof SEQ ID NO: 135 with a mutation selected from at least one of E46D,E46H, E46G, E46A, E46R, and E46K. In some embodiments, the capturedomain comprises the RNA binding domain of Tis11d having an amino acidsequence of SEQ ID NO: 136 with at least one mutation selected fromE27D, E27H, E27G, E27A, E27R, and E27K.

In some embodiments, a capture domain comprising Tis11d, or a fragmentor derivative thereof, comprises the amino acid sequence of SEQ ID NOs:134-136 with at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25 or more amino acid mutations.In some embodiments, the capture domain comprising Tis11d, or a fragmentor derivative thereof, comprises a sequence with at least 90%, at least95%, at least 97 %, or at least 99 % identity to any one of SEQ ID NOs:134-136.

In some embodiments, the capture domain comprises eukaryotic translationinitiation factor 4E (eIF4E), or a fragment or derivative thereof. eIF4E(see, e.g., Uniprot Accession No. P06730) binds to the mRNA cap. Theamino acid sequence of eIF4E is:

(M)ATVEPETTPTPNPPTTEEEKTESNQEVANPEHYIKHPLQNRWALWFFKNDKSKTWQANLRLISKFDTVEDFWALYNHIQLSSNLMPGCDYSLFKDGIEPMWEDEKNKRGGRWLITLNKQQRRSDLDRFWLETLLCLIGESFDDYSDDVCGAVVNVRAKGDKIAIWTTECENREAVTHIGRVYKERLGLPPKIVIGYQSHADTATKSGSTTKNRFVV (SEQ ID NO: 137).

In some embodiments, the capture domain comprises an eIF4E fragmenthaving an amino acid sequence of SEQ ID NO: 138.

In some embodiments, a capture domain comprising eIF4E, or a fragment orderivative thereof, comprises the amino acid sequence of SEQ ID NOs:137-138 with at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25 or more amino acid mutations.In some embodiments, the capture domain comprising eIF4E, or fragment orderivative thereof, comprises a sequence with at least 90%, at least95%, at least 97 %, or at least 99 % identity to any one of SEQ ID NOs:137-138.

In some embodiments, the capture domain comprises poly(A)-bindingprotein (PABP), or a fragment or derivative thereof. PABP (see, e.g.,Uniprot Accession No. P11940) binds to the poly(A) tail of mRNA. Theamino acid sequence of PABP is:

(M)NPSAPSYPMASLYVGDLHPDVTEAMLYEKFSPAGPILSIRVCRDMITRRSLGYAYVNFQQPADAERALDTMNFDVIKGKPVRIMWSQRDPSLRKSGVGNIFIKNLDKSIDNKALYDTFSAFGNILSCKVVCDENGSKGYGFVHFETQEAAERAIEKMNGMLLNDRKVFVGRFKSRKEREAELGARAKEFTNVYIKNFGEDMDDERLKDLFGKFGPALSVKVMTDESGKSKGFGFVSFERHEDAQKAVDEMNGKELNGKQIYVGRAQKKVERQTELKRKFEQMKQDRITRYQGVNLYVKNLDDGIDDERLRKEFSPFGTITSAKVMMEGGRSKGFGFVCFSSPEEATKAVTEMNGRIVATKPLYVALAQRKEERQAHLTNQYMQRMASVRAVPNPVINPYQPAPPSGYFMAAIPQTQNRAAYYPPSQIAQLRPSPRWTAQGARPHPFQNMPGAIRPAAPRPPFSTMRPASSQVPRVMSTQRVANTSTQTMGPRPAAAAAAATPAVRTVPQYKYAAGVRNPQQHLNAQPQVTMQQPAVHVQGQEPLTASMLASAPPQEQKQMLGERLFPLIQAMHPTLAGKITGMLLEIDNSELLHMLESPESLRSKVDEAVAVLQAHQAKEAAQKAVNSATGVPTV(SEQ ID NO:  139).

In some embodiments, the capture domain comprises a PABP fragment havingan amino acid sequence of SEQ ID NO: 140. In some embodiments, thecapture domain comprises the RNA recognition motif (RRM) 1 domain ofPABP having an amino acid sequence of SEQ ID NO: 141. In someembodiments, the capture domain comprises the RRM2 domain of PABP havingan amino acid sequence of SEQ ID NO: 142. In some embodiments, thecapture domain comprises the RRM3 domain of PABP having an amino acidsequence of SEQ ID NO: 143. In some embodiments, the capture domaincomprises the RRM4 domain of PABP having an amino acid sequence of SEQID NO: 144.

In some embodiments, a capture domain comprising PABP, or a fragment orderivative thereof, comprises the amino acid sequence of any one of SEQID NOs: 139-144 with at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25 or more amino acidmutations. In some embodiments, the capture domain comprising PABP, orfragment or derivative thereof, comprises a sequence with at least 90%,at least 95%, at least 97 %, or at least 99 % identity to any one of SEQID NOs: 139-144.

In some embodiments, the capture domain comprises Z-DNA binding protein1 (ZBP1), or a fragment or derivative thereof. ZBP1 (see, e.g., UniprotAccession No. Q9H171) binds to double stranded DNA. The amino acidsequence of ZBP1 is:

MAQAPADPGREGHLEQRILQVLTEAGSPVKLAQLVKECQAPKRELNQVLYRMKKELKVSLTSPATWCLGGTDPEGEGPAELALSSPAERPQQHAATIPETPGPQFSQQREEDIYRFLKDNGPQRALVIAQALGMRTAKDVNRDLYRMKSRHLLDMDEQSKAWTIYRPEDSGRRAKSASIIYQHNPINMICQNGPNSWISIANSEAIQIGHGNIITRQTVSREDGSAGPRHLPSMAPGDSSTWGTLVDPWGPQDIHMEQSILRRVQLGHSNEMRLHGVPSEGPAHIPPGSPPVSATAAGPEASFEARIPSPGTHPEGEAAQRIHMKSCFLEDATIGNSNKMSISPGVAGPGGVAGSGEGEPGEDAGRRPADTQSRSHFPRDIGQPITPSHSKLTPKLETMTLGNRSHKAAEGSHYVDEASHEGSWWGGGI (SEQ ID NO: 145).

In some embodiments, the capture domain comprises Z-binding domain 1 ofZBP1 having an amino acid sequence of SEQ ID NO: 146. In someembodiments, the capture domain comprises Z-binding domain 2 of ZBP1having an amino acid sequence of SEQ ID NO: 147.

In some embodiments, a capture domain comprising ZBP1, or a fragment orderivative thereof, comprises the amino acid sequence of any one of SEQID NOs: 145-147 with at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25 or more amino acidmutations. In some embodiments, the capture domain comprising ZBP1, orfragment or derivative thereof, comprises a sequence with at least 90%,at least 95%, at least 97 %, or at least 99 % identity to any one of SEQID NOs: 145-147.

In some embodiments, the capture domain comprises PUM-HDdomain-containing protein (PUM-HD), or a fragment or derivative thereof.PUM-HD (see, e.g., Uniprot Accession No. B2BXX4) binds to mRNA. Theamino acid sequence of PUM-HD is:

(M)HRGNEDLSFGDDYEKEIGLLLGEQQRRQEEADEIEKELNLYRSGSAPPTVDGSVNAAGGLFNGGGRGPFMEFGGGNKGNGFGGDDDELRKDPAYLSYYYANMKLNPRLPPPLMSREDLRVAQRLKGSSNVLGGVGDRRNVNESRSLFSMPPGFDQMNEFEAEKTNASSSEWDANGLIGLPGLGLGGKQKSFADIFQPDMGHPVSQQPSRPASRNAFDENVDSTNNQSPSASQGIGAPPPYSYAAVLGSSLSRNGTPDPQAVARVPSPCLTPIGSGRVSSNDKRNTSNQSPFNGVTSGLNESSDLVNALSGMNLSGSGGLDDRGQAEQDVEKVRNYMFGFQGGHNEVSQHVFPNKSDQAQKATGSLRNLHMRGSQGSAYNGGGLANPYQHLDSPNYCLNNYALNPAVASVMANQLGNSNFSPMYDNYSAASALGFSGMDSRLHGGGFESRNLGRSNRMMGGGGLQSHMADPMYHQYGRYSENVDALDLLNDPAMDRSFMGNSYMNMLELQRAYLGAQKSQYGVPYKSGSPNSHSYYGSPTFGSNMSYPGSPLAHHAMQNSLMSPCSPMRRGEVNMRYPSATRNYSGGVMGSWHMDASLDEGFGSSLLEEFKSNKTRGFELSEIAGHVVEFSADQYGSRFIQQKLETATTDEKNMVYEEIMPHALALMTDVFGNYVIQKFFEHGLPPQRRELGDKLFENVLPLSLQMYGCRVIQKAIEVVDLDQKIKMVKELDGHVMRCVRDQNGNHVVQKCIECVPEENIEFIISTFFGHVVSLSTHPYGCRVIQRVLEHCHDPDTQSKVMEEILSTVSMLAQDQYGNYVVQHVLEHGKPDERTVIIKELAGKIVQMSQQKFASNVVEKCLTFGGPEERELLVNEMLGTTDENEPLQAMMKDQFANYVVQKVLETCDDQQRELILTRIKVHLNALKKYTYGKHVVARIEKLVAAGGMHMFLLFPLGLKEENGFAVPNPASDVVRPQVLYSLTRVDGSAIAF (SEQ I D NO: 148).

In some embodiments, a capture domain comprising PUM-HD, or a fragmentor derivative thereof, comprises the amino acid sequence of SEQ ID NO:148 with at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25 or more amino acid mutations.In some embodiments, the capture domain comprising PUM-HD, or fragmentor derivative thereof, comprises a sequence with at least 90%, at least95%, at least 97 %, or at least 99 % identity to SEQ ID NO: 148.

In some embodiments, the capture domain binds a virus. In someembodiments, the virus is an adenovirus, an adeno-associated virus(AAV), a lentivirus, a retrovirus, a poxvirus or a herpesvirus. In someembodiments, the virus is a wildtype or mutant AAV selected from AAV1,AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74,AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV,snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAVAnc80, AAV PHP.B, and any other AAV now known or later discovered. Insome embodiments, the capture domain that binds to an AAV particle hasan amino acid sequence of SEQ ID NO: 166, or an amino acid sequence withat least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % identity to SEQ ID NO: 166.

In some embodiments, the capture domain binds an adenovirus particle.Adenoviruses are medium-sized (90-100 nm) nonenveloped viruses from thefamily Adenoviridae with an icosahedral nucleocapsid containing a doublestranded DNA genome. There are four genera of the family Adenoviridae(e.g., Aviadenovirus, Mastadenovirus, Atadenovirus, Ichtadenovirus,Siadenovirus). In some embodiments, the capture domain binds to anadenovirus from Aviadenovirus, Mastadenovirus, Atadenovirus,Ichtadenovirus, or Siadenovirus. In some embodiments, the capture domainbinds to an adenovirus from Mastadenovirus. Human adenoviruses aregrouped into seven different adenovirus serotypes: A, B, C, D, E, F, G,and H. In some embodiments, the capture domain binds to a humanadenovirus particle of serotypes A, B, C, D, E, F, G, or H.

The adenovirus capsid comprises three major types of proteins: hexon,penton base, and fiber. The coxsackievirus and adenovirus receptor (see,e.g., Uniprot Accession No. P78310), which is expressed on heart, brainepithelial, and endothelial cells, binds to the fiber protein of theadenovirus capsid. The amino acid sequence of the coxsackievirus andadenovirus receptor from Homo sapiens is:

(M)ALLLCFVLLCGVVDFARSLSITTPEEMIEKAKGETAYLPCKFTLSPEDQGPLDIEWLISPADNQKVDQVIILYSGDKIYDDYYPDLKGRVHFTSNDLKSGDASINVTNLQLSDIGTYQCKVKKAPGVANKKIHLVVLVKPSGARCYVDGSEEIGSDFKIKCEPKEGSLPLQYEWQKLSDSQKMPTSWLAEMTSSVISVKNASSEYSGTYSCTVRNRVGSDQCLLRLNVVPPSNKAGLIAGAIIGTLLALALIGLIIFCCRKKRREEKYEKEVHHDIREDVPPPKSRTSTARSYIGSNHSSLGSMSPSNMEGYSKTQYNQVPSEDFERTPQSPTLPPAKVAAPNLSRMGAIPVMIPAQSKDGSIV (SEQ ID NO: 62).

In some embodiments, a capture domain of a purification matrix providedherein comprises the coxsackievirus and adenovirus receptor, or afragment or derivative thereof.

In some embodiments, the capture domain comprising a coxsackievirus andadenovirus receptor, or fragment thereof, comprises an amino acidsequence selected from any one of SEQ ID NOs: 62-72. In someembodiments, the capture domain comprising a coxsackievirus andadenovirus receptor, or fragment thereof, comprises the amino acidsequence of any one of SEQ ID NOs: 62-72 with at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25 or more amino acid mutations. In some embodiments, the capture domaincomprising a coxsackievirus and adenovirus receptor, or fragmentthereof, comprises a sequence with at least 90%, at least 95%, at least97 %, or at least 99 % identity to any one of SEQ ID NOs: 62-72.

In some embodiments, the capture domain comprises the extracellulardomain of the coxsackievirus and adenovirus receptor, or a fragmentthereof. In some embodiments, the capture domain comprising theextracellular domain of the coxsackievirus and adenovirus receptor hasan amino acid sequence of SEQ ID NO: 63 or 64. In some embodiments, thecapture domain comprises domain 1 of the coxsackievirus and adenovirusreceptor, or a fragment thereof. In some embodiments, the capture domaincomprising domain 1 of the coxsackievirus and adenovirus receptor has anamino acid sequence of SEQ ID NO: 65. In some embodiments, the capturedomain comprises domain 2 of the coxsackievirus and adenovirus receptor,or a fragment thereof. In some embodiments, the capture domaincomprising domain 2 of the coxsackievirus and adenovirus receptor has anamino acid sequence of SEQ ID NO: 66. In some embodiments, the capturedomain comprises isoform 3 of the coxsackievirus and adenovirusreceptor, or a fragment thereof. In some embodiments, the capture domaincomprising isoform 3 of the coxsackievirus and adenovirus receptor hasan amino acid sequence of SEQ ID NO: 67. In some embodiments, thecapture domain comprises isoform 4 of the coxsackievirus and adenovirusreceptor, or a fragment thereof. In some embodiments, the capture domaincomprising isoform 4 of the coxsackievirus and adenovirus receptor hasan amino acid sequence of SEQ ID NO: 68. In some embodiments, thecapture domain comprises isoform 5 of the coxsackievirus and adenovirusreceptor, or a fragment thereof. In some embodiments, the capture domaincomprising isoform 5 of the coxsackievirus and adenovirus receptor hasan amino acid sequence of SEQ ID NO: 69. In some embodiments, thecapture domain comprises isoform 7 of the coxsackievirus and adenovirusreceptor, or a fragment thereof. In some embodiments, the capture domaincomprising isoform 7 of the coxsackievirus and adenovirus receptor hasan amino acid sequence of SEQ ID NO: 70.

In some embodiments, the capture domain comprises an amino acid sequenceof M(RAIVFRVQWLRRYFVNGSRSGGG)_(n), where n is an integer from 1 to 8(SEQ ID NO: 71), for example, n is 1, 2, 3, 4, 5, 6, 7, or 8. In someembodiments, the capture domain comprises an amino acid sequence of(RAIVFRVQWLRRYFVNGSRSGGG)_(n), wherein n is an integer from 1 to 8 (SEQID NO: 72), for example, 1, 2, 3, 4, 5, 6, 7, or 8.

In some embodiments, the capture domain that binds to an adenovirusparticle has an amino acid sequence of SEQ ID NO: 167, or an amino acidsequence with at least 90 %, at least 95 %, at least 96 %, at least 97%, at least 98 %, or at least 99 % identity to SEQ ID NO: 167.

In some embodiments, a capture domain of a purification matrix providedherein comprises the cluster of differentiation 80 (CD80), or a fragmentor derivative thereof. CD80 (see, e.g., Uniprot Accession No. P33681) isa type 1 membrane protein of the immunoglobulin superfamily. CD80 bindsto at least the knob domain of the fiber protein of adenovirus speciesB. The amino acid sequence of CD80 from Homo sapiens is

(M)GHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID N O: 98).

In some embodiments, the capture domain comprises the extracellulardomain of CD80, or a fragment or derivative thereof, having an aminoacid sequence of SEQ ID NO: 99 or SEQ ID NO: 100.

In some embodiments, the capture domain comprising CD80, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 98-100. In some embodiments, the capture domaincomprising CD80, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID NOs: 98-100 with at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising CD80, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID Nos: 98-100.

In some embodiments, a capture domain of a purification matrix providedherein comprises the cluster of differentiation 86 (CD86), or a fragmentor derivative thereof. CD86 (see, e.g., Uniprot Accession No. P42081) isa type 1 membrane protein of the immunoglobulin superfamily. CD86 bindsto at least the knob domain of the fiber protein of adenovirus speciesB. The amino acid sequence of CD86 from Homo sapiens is

(M)DPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF (SEQ ID NO: 101).

In some embodiments, the capture domain comprises the extracellulardomain of CD86. In some embodiments, the capture domain comprises theextracellular domain of CD86, having an amino acid sequence of SEQ IDNO: 102. In some embodiments, the capture domain comprises a fragment ofthe extracellular domain of CD86, having an amino acid sequence of SEQID NOs: 103 or 104.

In some embodiments, the capture domain comprising CD86, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 101-104. In some embodiments, the capture domaincomprising CD86, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID NOs: 101-104 with at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising CD86, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID Nos: 101-104.

In some embodiments, the capture domain binds a lentovirus particle.Lentivirus is a genus of retroviruses that causes chronic and deadlydiseases characterized by long incubation periods. Examples oflentiviruses include human immunodeficiency virus (HIV) and vesicularstomatitis virus (VSV). There are five serogroups of lentivirus: bovine,equine, feline, ovinecaprine, and primate. In some embodiments, thecapture domain binds a lentovirus particle of the bovine, equine,feline, ovinecaprine, or primate serogroups. In some embodiments,lentiviruses infect host cells via binding to the low-densitylipoprotein receptor (LDLR) or cluster of differentiation 4 (CD4).

In some embodiments, the capture domain that binds to an lentivirusparticle has an amino acid sequence of SEQ ID NO: 168, or an amino acidsequence with at least 90 %, at least 95 %, at least 96 %, at least 97%, at least 98 %, or at least 99 % identity to SEQ ID NO: 168. In someembodiments, the capture domain that binds to an lentivirus particle hasan amino acid sequence of SEQ ID NO: 169, or an amino acid sequence withat least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % identity to SEQ ID NO: 169.

In some embodiments, a capture domain of a purification matrix providedherein comprises the LDLR, or a fragment or derivative thereof. LDLR(see, e.g., Uniprot Accession No. P01130) is a cell-surface receptorthat mediates the endocytosis of cholesterol rich low-densitylipoprotein. The amino acid sequence of LDLR from Homo sapiens is

(M)GPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISYKWVCDGSAECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLVFLCLGVFLLWKNWRLKNINSINFDNPVYQKTTEDEVHICHNQDGYSYPSRQMVSLEDDVA (SEQ ID NO: 73).

In some embodiments, the capture domain comprises the extracellulardomain of LDLR having an amino acid sequence of SEQ ID NO: 74. In someembodiments, the capture domain comprises a fragment of theextracellular domain of LDLR having an amino acid sequence of SEQ ID NO:77. In some embodiments, the capture domain comprises the CR2 domain ofLDLR having an amino acid sequence of SEQ ID NO: 75. In someembodiments, the capture domain comprises the CR3 domain of LDLR havingan amino acid sequence of SEQ ID NO: 76.

In some embodiments, the capture domain comprising LDLR, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 73-76. In some embodiments, the capture domaincomprising LDLR, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID NOs: 73-76 with at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising LDLR, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID Nos: 73-76.

In some embodiments, a capture domain of a purification matrix providedherein comprises CD4, or a fragment or derivative thereof. In someembodiments, a capture domain comprising CD4 binds to a lentivirusparticle or a retrovirus particle. In some embodiments, CD4 binds toglycoprotein 120 (gp120) of human immunodeficiency virus.

CD4 (see, e.g., Uniprot Accession No. P01730) is a glycoprotein found onthe surface of immune cells such as T helper cells, monocytes,macrophages, and dendritic cells. The amino acid sequence of CD4 fromHomo sapiens is

(M)NRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI (SEQ ID NO: 78).

In some embodiments, the capture domain comprises CD4 having an aminoacid sequence of SEQ ID NO: 78. In some embodiments, the capture domaincomprises CD4 having an amino acid sequence of SEQ ID NO: 78 having atleast one, at least two, at least three, or at least four mutations ofamino acids 112, 113, 116, and 117 to glycine, alanine, lysine,arginine, or histidine.

In some embodiments, the capture domain comprises the extracellulardomain of CD4. In some embodiments, the capture domain comprises theextracellular domain of CD4 having an amino acid sequence of SEQ ID NO:79.

In some embodiments, the capture domain comprises a fragment of theextracellular domain of CD4 having an amino acid sequence of SEQ ID NO:80. In some embodiments, the capture domain comprises domain 1 of CD4having an amino acid sequence of SEQ ID NO: 81. In some embodiments, thecapture domain comprises the extracellular domain of CD4 or a fragmentor derivative thereof (SEQ ID NOs: 79-81) having at least one, at leasttwo, at least three, or at least four mutations at amino acids 88, 89,92, and 93 to glycine, alanine, lysine, arginine, or histidine.

In some embodiments, the capture domain comprising CD4, or a fragment orderivative thereof, comprises an amino acid sequence selected from anyone of SEQ ID NOs: 78-81. In some embodiments, the capture domaincomprising CD4, or a fragment or derivative thereof, comprises the aminoacid sequence of any one of SEQ ID NOs: 78-81 with at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7, at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25 or more amino acid mutations. In some embodiments, the capturedomain comprising CD4, or fragment thereof, comprises a sequence with atleast 90%, at least 95%, at least 97 %, or at least 99 % identity to anyone of SEQ ID Nos: 78-81.

In some embodiments, a capture domain of a purification matrix providedherein comprises cluster of differentiation 46 (CD46), or a fragment orderivative thereof. In some embodiments, a capture domain comprisingCD46 or a fragment or derivative thereof binds to a measles virusparticle, a herpesvirus particle, an adenovirus particle, Streptococcuspyogenes, or pathogenic Nesseria. CD46 (see, e.g., Uniprot Accession No.P15529) is a type 1 membrane protein and is a regulatory part of thecomplement system. In some embodiments, a capture domain comprising CD46or a fragment or derivative thereof binds to group B adenoviruses. Insome embodiments, a capture domain comprising CD46 or a fragment orderivative thereof binds to human herpesvirus-6. The amino acid sequenceof CD46 is:

(M)EPPGRRECPFPSWRFPGLLLAAMVLLLYSFSDACEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHSVSTSSTTKSPASSASGPRPTYKPPVSNYPGYPKPEEGILDSLDVWVIAVIVIAIVVGVAVICVVPYRYLQRRKKKGTYLTDETHREVKFTSL (SEQ  ID NO: 82).

In some embodiments, a capture domain comprises the extracellular domainof CD46 or a fragment or derivative thereof. In some embodiments, acapture domain comprises the extracellular domain of CD46 or a fragmentor derivative thereof, having an amino acid sequence of SEQ ID NO: 83 orSEQ ID NO: 84. In some embodiments, the capture domain comprises domain1 of CD46 having an amino acid sequence of SEQ ID NO: 85. In someembodiments, the capture domain comprises domain 2 of CD46 having anamino acid sequence of SEQ ID NO: 86. In some embodiments, the capturedomain comprises domain 3 of CD46 having an amino acid sequence of SEQID NO: 87. In some embodiments, the capture domain comprises domain 4 ofCD46 having an amino acid sequence of SEQ ID NO: 88. In someembodiments, the capture domain comprises domain 5 of CD46 having anamino acid sequence of SEQ ID NO: 89.

In some embodiments, the capture domain comprising CD46, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 82-89. In some embodiments, the capture domaincomprising CD46, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID NOs: 82-89 with at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising CD46, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID Nos: 82-89.

In some embodiments, the capture domain binds a measles virus particle.Measles virus is a single-stranded, negative-sense, enveloped,non-segmented RNA virus of the genus Morbillivirus within the familyParamyxoviridae. The measles virus has two envelope glycoproteins on theviral surface: hemagglutinin (H) and membrane fusion protein (F).Receptors for the measles H protein include CD46, the signalinglymphocyte activation molecule (SLAMF1), and the cell adhesion moleculeNectin-4.

In some embodiments, a capture domain of a purification matrix providedherein comprises SLAMF1, or a fragment or derivative thereof. In someembodiments, a capture domain comprising SLAMF1 or a fragment orderivative thereof binds to a measles virus particle. SLAMF1 (see, e.g.,Uniprot Accession No. Q13291) belongs to the signaling lymphocyticactivation molecule family. The amino acid sequence of SLAMF1 is:

(M)DPKGLLSLTFVLFLSLAFGASYGTGGRMMNCPKILRQLGSKVLLPLTYERINKSMNKSIHIVVTMAKSLENSVENKIVSLDPSEAGPPRYLGDRYKFYLENLTLGIRESRKEDEGWYLMTLEKNVSVQRFCLQLRLYEQVSTPEIKVLNKTQENGTCTLILGCTVEKGDHVAYSWSEKAGTHPLNPANSSHLLSLTLGPQHADNIYICTVSNPISNNSQTFSPWPGCRTDPSETKPWAVYAGLLGGVIMILIMVVILQLRRRGKTNHYQTTVEKKSLTIYAQVQKPGPLQKKLDSFPAQDPCTTIYVAATEPVPESVQETNSITVYASVTLPES (SEQ ID NO:  90).

In some embodiments, a capture domain comprises the extracellular domainof SLAMF1 or a fragment or derivative thereof. In some embodiments, acapture domain comprises the extracellular domain of SLAMF1 or afragment or derivative thereof, having an amino acid sequence of SEQ IDNO: 91 or SEQ ID NO: 92.

In some embodiments, the capture domain comprising SLAMF1, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 90-92. In some embodiments, the capture domaincomprising SLAMF1, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID NOs: 90-92 with at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising SLAMF1, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID Nos: 90-92.

In some embodiments, a capture domain of a purification matrix providedherein comprises Nectin-4, or a fragment or derivative thereof. In someembodiments, a capture domain comprising Nectin-4 or a fragment orderivative thereof binds to a measles virus particle. Nectin-4 (see,e.g., Uniprot Accession No. Q96NY8) belongs to the family of cellularadhesion molecules involved in calcium-independent cellular adhesion.The amino acid sequence of Nectin-4 is:

(M)PLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 93).

In some embodiments, a capture domain comprises the extracellular domainof Nectin-4 or a fragment or derivative thereof. In some embodiments, acapture domain comprises the extracellular domain of Nectin-4 or afragment or derivative thereof, having an amino acid sequence of SEQ IDNO: 94. In some embodiments, a capture domain comprises domain 1 ofNectin-4 or a fragment or derivative thereof, having an amino acidsequence of SEQ ID NO: 95. In some embodiments, a capture domaincomprises domain 2 of Nectin-4 or a fragment or derivative thereof,having an amino acid sequence of SEQ ID NO: 96. In some embodiments, acapture domain comprises domain 3 of Nectin-4 or a fragment orderivative thereof, having an amino acid sequence of SEQ ID NO: 97.

In some embodiments, the capture domain comprising Nectin-4, or afragment or derivative thereof, comprises an amino acid sequenceselected from any one of SEQ ID NOs: 93-97. In some embodiments, thecapture domain comprising Nectin-4, or a fragment or derivative thereof,comprises the amino acid sequence of any one of SEQ ID NOs: 93-97 withat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25 or more amino acid mutations. In someembodiments, the capture domain comprising Nectin-4, or fragmentthereof, comprises a sequence with at least 90%, at least 95%, at least97 %, or at least 99 % identity to any one of SEQ ID Nos: 93-97.

In some embodiments, the capture domain binds a baboon endogenous virusparticle. Baboon endogenous virus (BaEV) is a type C endogenousoncovirus. BaEV contains a type D envelope (env) gene. BaEV virusparticles bind to neutral amino acid transporter B(0) (SLC1A5) and/orneutral amino acid transporter A (SLC1A4).

In some embodiments, a capture domain of a purification matrix providedherein comprises SLC1A5, or a fragment or derivative thereof. SLC1A5(see, e.g., Uniprot Accession No. Q15758) is a sodium-dependent aminoacid transporter. The amino acid sequence of SLCIA5 is:

(M)VADPPRDSKGLAAAEPTANGGLALASIEDQGAAAGGYCGSRDQVRRCLRANLLVLLTVVAVVAGVALGLGVSGAGGALALGPERLSAFVFPGELLLRLLRMIILPLVVCSLIGGAASLDPGALGRLGAWALLFFLVTTLLASALGVGLALALQPGAASAAINASVGAAGSAENAPSKEVLDSFLDLARNIFPSNLVSAAFRSYSTTYEERNITGTRVKVPVGQEVEGMNILGLVVFAIVFGVALRKLGPEGELLIRFFNSFNEATMVLVSWIMWYAPVGIMFLVAGKIVEMEDVGLLFARLGKYILCCLLGHAIHGLLVLPLIYFLFTRKNPYRFLWGIVTPLATAFGTSSSSATLPLMMKCVEENNGVAKHISRFILPIGATVNMDGAALFQCVAAVFIAQLSQQSLDFVKIITILVTATASSVGAAGIPAGGVLTLAIILEAVNLPVDHISLILAVDWLVDRSCTVLNVEGDALGAGLLQNYVDRTESRSTEPELIQVKSELPLDPLPVPTEEGNPLLKHYRGPAGDATVASEKESVM (SEQ I D NO: 105).

In some embodiments, a capture domain comprises the extracellular domainof SLC1A5 or a fragment or derivative thereof. In some embodiments, acapture domain comprises the extracellular domain of SLC1A5 or afragment or derivative thereof, having an amino acid sequence of any oneof SEQ ID NOs: 106-110.

In some embodiments, the capture domain comprising SLC1A5, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 105-110. In some embodiments, the capture domaincomprising SLC1A5, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID NOs: 105-110 with at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising SLC1A5, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID NOs: 105-110.

In some embodiments, a capture domain of a purification matrix providedherein comprises SLC1A4, or a fragment or derivative thereof. SLC1A4(see, e.g., Uniprot Accession No. P43007) is a transporter of alanine,serine, cysteine, and threonine. The amino acid sequence of SLC1A4 is:

(M)EKSNETNGYLDSAQAGPAAGPGAPGTAAGRARRCAGFLRRQALVLLTVSGVLAGAGLGAALRGLSLSRTQVTYLAFPGEMLLRMLRMIILPLVVCSLVSGAASLDASCLGRLGGIAVAYFGLTTLSASALAVALAFIIKPGSGAQTLQSSDLGLEDSGPPPVPKETVDSFLDLARNLFPSNLVVAAFRTYATDYKVVTQNSSSGNVTHEKIPIGTEIEGMNILGLVLFALVLGVALKKLGSEGEDLIRFFNSLNEATMVLVSWIMWYVPVGIMFLVGSKIVEMKDIIVLVTSLGKYIFASILGHVIHGGIVLPLIYFVFTRKNPFRFLLGLLAPFATAFATCSSSATLPSMMKCIEENNGVDKRISRFILPIGATVNMDGAAIFQCVAAVFIAQLNNVELNAGQIFTILVTATASSVGAAGVPAGGVLTIAIILEAIGLPTHDLPLILAVDWIVDRTTTVVNVEGDALGAGILHHLNQKATKKGEQELAEVKVEAIPNCKSEEETSPLVTHQNPAGPVASAPELESKESVL (SEQ IDNO: 111) .

In some embodiments, a capture domain comprises the extracellular domainof SLC1A4 or a fragment or derivative thereof. In some embodiments, acapture domain comprises the extracellular domain of SLC1A4 or afragment or derivative thereof, having an amino acid sequence of SEQ IDNO: 112. In some embodiments, a capture domain comprises a fragment ofSLC1A4 or a fragment or derivative thereof, having an amino acidsequence of SEQ ID NO: 113.

In some embodiments, the capture domain comprising SLC1A4, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 111-113. In some embodiments, the capture domaincomprising SLC1A4, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID NOs: 111-113 with at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising SLC1A4, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID NOs: 111-113.

In some embodiments, the capture domain binds a Nipah or Hendra virusparticle. Nipah and Hendra viruses are both members of theParamyxoviridae family and cause respiratory and nervous system disease.Nipah and Hendra viruses enter cells via the ephrin-B2 and ephrin-B3.

In some embodiments, a capture domain of a purification matrix providedherein comprises ephrin-B2, or a fragment or derivative thereof. In someembodiments, a capture domain comprising ephrin-B2 or a fragment orderivative thereof binds to a Nipah virus particle or a Hendra virusparticle. Ephrin-B2 (see, e.g., Uniprot Accession No. P52799) bindspromiscuously to ephrin receptors. Ephrin receptors are a family ofreceptor tyrosine kinases which are crucial for migration, repulsion,and adhesion during neuronal, vascular, and epithelial development. Theamino acid sequence of ephrin-B2 is:

(M)AVRRDSVWKYCWGVLMVLCRTAISKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVGQDASSAGSTRNKDPTRRPELEAGTNGRSSTTSPFVKPNPGSSTDGNSAGHSGNNILGSEVALFAGIASGCIIFIVIIITLVVLLLKYRRRHRKHSPQHTTTLSLSTLATPKRSGNNNGSEPSDIIIPLRTADSVFCPHYEKVSGDYGHPVYIVQEMPPQSPANIYYKV (SEQ ID NO: 11 4).

In some embodiments, a capture domain comprises the extracellular domainof ephrin-B2 or a fragment or derivative thereof. In some embodiments, acapture domain comprises the extracellular domain of ephrin-B2 or afragment or derivative thereof, having an amino acid sequence of SEQ IDNO: 115.

In some embodiments, the capture domain comprising ephrin-B2, or afragment or derivative thereof, comprises an amino acid sequenceselected from SEQ ID NO: 114 or SEQ ID NO: 115. In some embodiments, thecapture domain comprising ephrin-B2, or a fragment or derivativethereof, comprises the amino acid sequence of SEQ ID NO: 114 or SEQ IDNO: 115 with at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25 or more amino acid mutations.In some embodiments, the capture domain comprising ephrin-B2, orfragment thereof, comprises a sequence with at least 90%, at least 95%,at least 97 %, or at least 99 % identity to SEQ ID NO: 114 or SEQ ID NO:115.

In some embodiments, the capture domain binds a retrovirus particle. Insome embodiments, the capture domain binds to Gibbon-ape leukemia virus(GaLV). GaLV is an oncogenic type C retrovirus isolated from thewhite-handed gibbon and wooly monkey. GaLV virus enters host cells viathe sodium-dependent phosphate transporter 1 (SLC20A1) orsodium-dependent phosphate transporter 2 (SLC20A2).

In some embodiments, a capture domain of a purification matrix providedherein comprises SLC20A1, or a fragment or derivative thereof. In someembodiments, a capture domain comprising SLC20A1 or a fragment orderivative thereof binds to a GaLV virus particle. SLC20A1 (see, e.g.,Uniprot Accession No. Q8WUM9) is a sodium-phosphate symporter, whichconfers human cells susceptibility to GaLV. The amino acid sequence ofSLC20A1 is:

MATLITSTTAATAASGPLVDYLWMLILGFIIAFVLAFSVGANDVANSFGTAVGSGVVTLKQACILASIFETVGSVLLGAKVSETIRKGLIDVEMYNSTQGLLMAGSVSAMFGSAVWQLVASFLKLPISGTHCIVGATIGFSLVAKGQEGVKWSELIKIVMSWFVSPLLSGIMSGILFFLVRAFILHKADPVPNGLRALPVFYACTVGINLFSIMYTGAPLLGFDKLPLWGTILISVGCAVFCALIVWFFVCPRMKRKIEREIKCSPSESPLMEKKNSLKEDHEETKLSVGDIENKHPVSEVGPATVPLQAVVEERTVSFKLGDLEEAPERERLPSVDLKEETSID STVNGAVQLPNGNLVQFSQAVSNQINSSGHYQYHTVHKDSGLYKELLHKLHLAKVGDCMGDSGDKPLRRNNSYTSYTMAICGMPLDSFRAKEGEQKGEEMEKLTWPNADSKKRIRMDSYTSYCNAVSDLHSASEIDMSVKAEMGLGDRKGSNGSLEEWYDQDKPEVSLLFQFLQILTACFGSFAHGGNDVSNAIGPLVALYLVYDTGDVSSKVATPIWLLLYGGVGICVGLWVWGRRVIQTMGKDLTPITPSSGFSIELASALTVVIASNIGLPISTTHCKVGSVVSVGWLRSKKAVDWRLFRNIFMAWFVTVPISGVISAAIMAIFRYVILRM (SEQ ID NO: 116).

In some embodiments, a capture domain comprises a fragment of SLC20A1,having an amino acid sequence of any one of SEQ ID NOs: 117 and 118. Insome embodiments, a capture domain comprises SLC20A1 or a fragment orderivative thereof, having an amino acid sequence of SEQ ID NO: 116 witha mutation of Asp-550 to lysine, arginine, glycine, alanine, orhistidine. In some embodiments, a capture domain comprises SLC20A1 or afragment or derivative thereof, having an amino acid sequence of SEQ IDNO: 117 with a mutation of Asp-20 to lysine, arginine, glycine, alanine,or histidine.

In some embodiments, the capture domain comprising SLC20A1, or afragment or derivative thereof, comprises an amino acid sequenceselected from any one of SEQ ID NOs: 116-118. In some embodiments, thecapture domain comprising SLC20A1, or a fragment or derivative thereof,comprises the amino acid sequence of any one of SEQ ID Nos: 116-118 withat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25 or more amino acid mutations. In someembodiments, the capture domain comprising SLC20A1, or fragment thereof,comprises a sequence with at least 90%, at least 95%, at least 97 %, orat least 99 % identity to any one of SEQ ID NOs: 116-118.

In some embodiments, a capture domain of a purification matrix providedherein comprises SLC20A2, or a fragment or derivative thereof. In someembodiments, a capture domain comprising SLC20A2or a fragment orderivative thereof binds to a GaLV virus particle. SLC20A2 (see, e.g.,Uniprot Accession No. Q08357) is a sodium-phosphate symporter, whichconfers human cells susceptibility to GaLV. The amino acid sequence ofSLC20A2 is:

(M)AMDEYLWMVILGFIIAFILAFSVGANDVANSFGTAVGSGVVTLRQACILASIFETTGSVLLGAKVGETIRKGIIDVNLYNETVETLMAGEVSAMVGSAVWQLIASFLRLPISGTHCIVGSTIGFSLVAIGTKGVQWMELVKIVASWFISPLLSGFMSGLLFVLIRIFILKKEDPVPNGLRALPVFYAATIAINVFSIMYTGAPVLGLVLPMWAIALISFGVALLFAFFVWLFVCPWMRRKITGKLQKEGALSRVSDESLSKVQEAESPVFKELPGAKANDDSTIPLTGAAGETLGTSEGTSAGSHPRAAYGRALSMTHGSVKSPISNGTFGFDGHTRSDGHVYHTVHKDSGLYKDLLHKIHIDRGPEEKPAQESNYRLLRRNNSYTCYTAAICGLPVHATFRAADSSAPEDSEKLVGDTVSYSKKRLRYDSYSSYCNAVAEAEIEAEEGGVEMKLASELADPDQPREDPAEEEKEEKDAPEVHLLFHFLQVLTACFGSFAHGGNDVSNAIGPLVALWLIYKQGGVTQEAATPVWLLFYGGVGICTGLWVWGRRVIQTMGKDLTPITPSSGFTIELASAFTVVIASNIGLPVSTTHCKVGSVVAVGWIRSRKAVDWRLFRNIFVAWFVTVPVAGLFSAAVMALLMYGI LPYV(SEQ ID NO: 119).

In some embodiments, a capture domain comprises the extracellular domainof SLC20A2, having an amino acid sequence of SEQ ID NO: 120. In someembodiments, a capture domain comprises SLC20A2 or a fragment orderivative thereof, having an amino acid sequence of SEQ ID NO: 119 witha mutation of Lys-522 to aspartate, glycine, alanine, glutamate, orhistidine. In some embodiments, a capture domain comprises SLC20A2 or afragment or derivative thereof, having an amino acid sequence of SEQ IDNO: 120 with a mutation of Lys-20 to aspartate, glycine, alanine,glutamate, or histidine.

In some embodiments, the capture domain comprising SLC20A2, or afragment or derivative thereof, comprises an amino acid sequenceselected from any one of SEQ ID NOs: 119-120. In some embodiments, thecapture domain comprising SLC20A2, or a fragment or derivative thereof,comprises the amino acid sequence of any one of SEQ ID Nos: 119-120 withat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25 or more amino acid mutations. In someembodiments, the capture domain comprising SLC20A2, or fragment thereof,comprises a sequence with at least 90%, at least 95%, at least 97 %, orat least 99 % identity to any one of SEQ ID NOs: 119-120.

In some embodiments, the capture domain binds a coronavirus particle. Insome embodiments, the capture domain binds to SARS-CoV-2 or SARS-CoV.SARS-CoV-2 and SARS-CoV enter host cells via angiotension-convertingenzyme 2 (ACE2).

In some embodiments, a capture domain of a purification matrix providedherein comprises ACE2, or a fragment or derivative thereof. In someembodiments, a capture domain comprising ACE2 or a fragment orderivative thereof binds to a SARS-CoV or SARS-CoV-2 virus particle.ACE2 (see, e.g., Uniprot Accession No. Q9BYF1) is a carboxypeptidase ofthe renin-angiotensin hormone system. The amino acid sequence of ACE2is:

(M)SSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF (SEQ ID NO: 121).

In some embodiments, a capture domain comprises the extracellular domainof ACE2 or a fragment thereof. In some embodiments, a capture domaincomprises the extracellular domain of ACE2 or a fragment thereof, havingan amino acid sequence of any one of SEQ ID NOs: 122-124. In someembodiments, a capture domain comprises ACE2 or a fragment or derivativethereof, having an amino acid sequence of SEQ ID NO: 121 with one ormore mutations selected from K419E, K419D, K419H, K419G, K419A, N89G,and N89A. In some embodiments, a capture domain comprises ACE2 or afragment or derivative thereof, having an amino acid sequence of SEQ IDNO: 121 with one or more mutations of K31, H34, E35, N90, E208, H374,H378 to any amino acid selected from arginine, lysine, glutamic acid,aspartic acid, histidine, alanine, glycine, serine, threonine, andtryptophan.

In some embodiments, a capture domain comprises ACE2 or a fragment orderivative thereof, having an amino acid sequence of SEQ ID NO: 122 withone or more mutations selected from K403E, K403D, K403H, K403G, K403A,N73G, and N73A. In some embodiments, a capture domain comprises ACE2 ora fragment or derivative thereof, having an amino acid sequence of SEQID NO: 123 with one or more mutations selected from K402E, K402D, K402H,K402G, K402A, N72G, and N72A. In some embodiments, a capture domaincomprises ACE2 or a fragment or derivative thereof, having an amino acidsequence of SEQ ID NO: 124 with a mutation of N72G or N72A.

In some embodiments, the capture domain comprising ACE2, or a fragmentor derivative thereof, comprises an amino acid sequence selected fromany one of SEQ ID NOs: 121-124. In some embodiments, the capture domaincomprising ACE2, or a fragment or derivative thereof, comprises theamino acid sequence of any one of SEQ ID Nos: 121-124 with at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25 or more amino acid mutations. In some embodiments, thecapture domain comprising ACE2, or fragment thereof, comprises asequence with at least 90%, at least 95%, at least 97 %, or at least 99% identity to any one of SEQ ID NOs: 121-124.

In some embodiments, the biologic has a size from about 0.001 µm toabout 500 µm in diameter or length. In some embodiments, the biologichas a diameter between 1 nm and 100 µm, inclusive of the endpoints. Insome embodiments, the biologic has a diameter between 1 nm and 100 nm,inclusive of the endpoints. In some embodiments, the biologic has adiameter between 100 nm and 1 µm, inclusive of the endpoints. In someembodiments, the biologic has a diameter between 1 µm and 50 µm,inclusive of the endpoints. In some embodiments, the biologic has adiameter between 50 µm and 100 µm, inclusive of the endpoints.

In some embodiments, the size (i.e., diameter or length) of the biologicis about 0.001 µm, about 0.002 µm, about 0.003 µm, about 0.004 µm, about0.005 µm, about 0.006 µm, about 0.007 µm, about 0.008 µm, about 0.009µm, about 0.010 µm, about 0.020 µm, about 0.030 µm, about 0.040 µm,about 0.050 µm, about 0.060 µm, about 0.070 µm, about 0.080 µm, about0.090 µm, about 0.1 µm, about 0.2 µm, about 0.3 µm, about 0.4 µm, about0.5 µm, about 0.6 µm, about 0.7 µm, about 0.8 µm, about 0.9 µm, about 1µm, about 2 µm, about 3 µm, about 4 µm, about 5 µm, about 6 µm, about 7µm, about 8 µm, about 9 µm, about 10 µm, about 11 µm, about 12 µm, about13 µm, about 14 µm, about 15 µm, about 16 µm, about 17 µm, about 18 µm,about 19 µm, about 20 µm, about 21 µm, about 22 µm, about 22 µm, about23 µm, about 24 µm, about 25 µm, about 26 µm, about 27 µm, about 28 µm,about 29 µm, about 30 µm, about 31 µm, about 32 µm, about 33 µm, about34 µm, about 35 µm, about 36 µm, about 37 µm, about 38 µm, about 39 µm,about 40 µm, about 41 µm, about 42 µm, about 43 µm, about 44 µm, about45 µm, about 46 µm, about 47 µm, about 48 µm, about 49 µm, about 50 µm,about 55 µm, about 60 µm, about 65 µm, about 70 µm, about 75 µm, about80 µm, about 85 µm, about 90 µm, about 95 µm, about 100 µm, about 150µm, about 200 µm, about 250 µm, about 300 µm, about 350 µm, about 400µm, about 450 µm, or about 500 µm, or greater, including all values andranges in between. In some embodiments, the biologic has a size ofgreater than or equal to 10 µm. In some embodiments, the biologic has asize that is greater than or equal to 25 µm. In some embodiments, thebiologic has a size that is greater than or equal to 50 µm. In someembodiments, the biologic has a size that is greater than or equal to100 µm.

In some embodiments, the biologic has a size (i.e., molar mass) fromabout 2 kDa to about 1000 MDa. In some embodiments, the biologic has amolar mass of about 2 kDa, about 5 kDa, about 15 kDa, about 20 kDa,about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa,about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa,about 70 kDa, about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa,about 95 kDa, about 100 kDa, about 150 kDa, about 200 kDa, about 250kDa, about 300 kDa, about 350 kDa, about 400 kDa, about 450 kDa, about500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 700 kDa,about 750 kDa, about 800 kDa, about 850 kDa, about 900 kDa, about 950kDa, about 1000 kDa, about 1 MDa, about 5 MDa, about 10 MDa, about 15MDa, about 20 MDa, about 25 MDa, about 50 MDa, about 75 MDa, about 100MDa, about 125 MDa, about 150 MDa, about 175 MDa, about 200 MDa, about225 MDa, about 250 MDa, about 275 MDa, about 300 MDa, about 325 MDa,about 350 MDa, about 400 MDa, about 425 MDa, about 450 MDa, about 500MDa, about 550 MDa, about 600 MDa, about 650 MDa, about 700 MDa, about750 MDa, about 800 MDa, about 850 MDa, about 900 MDa, about 950 MDa, orabout 1000 MDa, including all values and ranges therebetween.

In some embodiments, the capture domain comprises an amino acid sequenceselected from the group consisting of: (a)

VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKK LNDAQAPK (SEQ ID NO:24);

(b)

VDNKFNKEQQNAFYEILSLPNLNEEQRAAFIQSLKDDPSQSANLLAEAKK LNDAQAPKG (SEQ ID NO:25)

(c)

VDNKFNKEHQNAFYEILHLPNLNEEQRNAFIQSLKHDPSQSANLLAEAKK LNDAQAPKG (SEQ ID NO:26);

(d)

AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEIL HLPN (SEQ ID NO: 27);

(e)

TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKT FTVTEG (SEQ ID NO:28);

(f)

KTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEMVTEVPGDAPTEPEKPEASIPLVPLTPATPIAKDDAKKDDTKKEDAKKPEAKKDDAKKAET (SEQ ID NO : 29);

(g)

KEETPETPETDSEEEVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLN1KFAG (SEQ ID NO: 30);

(h)

GYVS(R/H/K)(R/H)(P/S) (SEQ ID NO: 31);

(i)

SDVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYTWHWIRQFPGNKQEWGYIHFSGYTNYNPSLKSRVSITRDTSKNQFFLHLNSVTTEDTATYYCARGDYGYEWFTYWGQGTLVTVSADIQMTQSSSSFSVSLGDRVTITCKASEDIHNRLAWYKQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQNEDVATYYCQQYWIGPFTFGSGTNLEIK (SEQID NO: 32)

(j)

GYVSRHPGGGC (SEQ ID NO: 33);

(k)

GYVSRHPGGGGS (SEQ ID NO: 34);

(l)

FHENWPSGGGC (SEQ ID NO: 35);

(m)

FHENWPSGGGGS (SEQ ID NO: 36);

(n)

GVVTINP (SEQ ID NO: 37);

(o)

GLVTPSG (SEQ ID NO: 38);

(p)

GYVSHRS (SEQ ID NO: 39);

(q)

KVWILTP (SEQ ID NO: 40);

(r)

KLWVIPQ (SEQ ID NO: 41)

(s)

GVSAGESVQITLPKNEVQLNAYVLQEPPKGETYTYDWQLITHPRDYSGEMEGKHSQILKLSKLTPGLYEFKVIVEGQNAHGEGYVNVTVKPEPRKNRPPIAIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQYHWEELKGPLREEKISEDTAILKLSKLVPGNYTFSLTVVDSDGATNSTTANLTVNKAVDYPPVANAGPNQVITLPQNSITLFGNQSTDDHGITSYEWSLSPSSKGKVVEMQGVRTPTLQLSAMQEGDYTYQLTVTDTIGQQATAQVTVIVQPENNKPPQADAGPDKELTLPVDSTTLDGSKSSDDQKIISYLWEKTQGPDGVQLENANSSVATVTGLQVGTYVFTLTVKDERNLQSQSSVNVIVKEEINKPPIAKITGNVVITLPTSTAELDGSKSSDDKGIVSYLWTRDEGSPAAGEVLNHSDHHPILFLSNLVEGTYTFHLKVTDAKGESDTDRTTVEVKPDPRG (SEQ ID NO: 42)

(t)

SAGESVQITLPKNEVQLNAYVLQEPPKGETYTYDWQLITHPRDYSGEMEGKHSQILKLSKLTPGLYEFKVIVEGQNAHGEGYVNVTVKPE (SEQ ID N O: 43)

(u)

IAIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQYHWEELKGPLREEKISEDTAILKLSKLVPGNYTFSLTVVDSDGATNSTTANLTVNKA (SEQ ID N O: 44)

(v)

MGVSAGESVQITLPKNEVQLNAYVLQEPPKGETYTYDWQLITHPRDYSGEMEGKHSQILKLSKLTPGLYEFKVIVEGQNAHGEGYVNVTVKPEPRKNRPPIAIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQYHWEELKGPLREEKISEDTAILKLSKLVPGNYTFSLTVVDSDGATNSTTANLTVNKA (SEQ ID N O: 45)

(w)

VANAGPNQVITLPQNSITLFGNQSTDDHGITSYEWSLSPSSKGKVVEMQGVRTPTLQLSAMQEGDYTYQLTVTDTIGQQATAQVTVIVQPE (SEQ ID NO: 46)

(x)

QADAGPDKELTLPVDSTTLDGSKSSDDQKIISYLWEKTQGPDGVQLENANSSVATVTGLQVGTYVFTLTVKDERNLQSQSSVNVIVKEE (SEQ ID NO : 47)

(y)

IAKITGNVVITLPTSTAELDGSKSSDDKGIVSYLWTRDEGSPAAGEVLNHSDHHPILFLSNLVEGTYTFHLKVTDAKGESDTDRTTVEVKPDP (SEQ I D NO: 48)

, and (z)

LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SE QID NO: 49).

In some embodiments, the capture domain comprises an amino acid sequenceselected from any one of SEQ ID Nos: 24-49,62-148, 167-171. In someembodiments, the capture domain comprises an amino acid sequence with atleast 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %,at least 99 % identity to any one of SEQ ID Nos: 24-49, 62-148, and167-171.

In some embodiments, the capture domain comprises an amino acid sequencelacking the N-terminal methionine of any one of SEQ ID Nos: 24-49,62-148, and 167-171.

In some embodiments, the capture domain binds a contaminant. In someembodiments, the contaminant is a biologic. In some embodiments, thecontaminant is selected from the group consisting of a solvent, anendotoxin, a protein, a peptide, a nucleic acid, a virus, and acarbohydrate.

In some embodiments, the capture domain comprises a signal peptide.

In some embodiments, the number of capture domains within aprotein-based purification matrix ranges from 1 to about 100. In someembodiments, the number of capture domains is about 1, about 5, about10, about 20, about 30, about 40, about 50, about 55, about 60, about65, about 70, about 75, about 80, about 85, about 90, about 95, or about100. In some embodiments, a single polypeptide with phase behavior maybe coupled to multiple capture domains, such as about 1 to 100 capturedomains.

In some embodiments, a protein-based purification matrix may have two ormore capture domains that each individually bind to different biologics,contaminants, or other molecules.

In some embodiments, the affinity of a capture domain for a biologic,contaminant, and/or other molecule is modulated to facilitate separationof the biologic from the protein-based purification matrix.

In some embodiments, the capture domain is selected from the groupconsisting of protein A, protein G, and protein L, or a fragment orderivative thereof. In some embodiments, the capture domain is anantibody or fragment thereof (e.g., a Fab). In some embodiments, theantibody fragment thereof is a single-chain variable fragment.

Polypeptides With Phase Behavior

The compositions and methods disclosed herein may employ one or morepolypeptides with phase behavior.

In some embodiments, the polypeptide with phase behavior is aresilin-like polypeptide (RLP). Resilin-like polypeptides areelastomeric polypeptides with mechanical properties including desirableresilience, compressive elastic modulus, tensile elastic modulus, shearmodulus, extension to break, maximum tensile strength, hardness,rebound, and compression set. In some embodiments, the resilin-likepolypeptides described herein are polymers which comprise one or morerepeats. In some embodiments, the polymeric repeats may have an aminoacid sequence selected from any one of SEQ ID NOS: 1-9.

In some embodiments, a resilin-like polypeptide comprises more than onetype of repeat, e.g. a repeat of SEQ ID NO: 1 and a repeat of SEQ ID NO:3.

In some embodiments, the resilin-like polypeptides described hereincomprise repeats that occur up to 500 times within a given RLP. In someembodiments, the repeats occur about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, about 9, about 10, about 20, about 30,about 40, about 50, about 60, about 70, about 80, about 90, about 100,about 110, about 120, about 130, about 140, about 150, about 160, about170, about 180, about 190, about 200, about 210, about 220, about 230,about 240, about 250, about 260, about 270, about 280, about 290, about300, about 310, about 320, about 330, about 340, about 350, about 360,about 370, about 380, about 390, about 400, about 450, or about 500times.

In some embodiments, the RLP comprises one or more partial repeats. Insome embodiments, the length of a partial repeat is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more amino acids. In some embodiments, the RLP comprisesone or more additional amino acids at the N-terminus or C-terminus ofthe RLP that are not part of a repeat.

In some embodiments, one or more RLP repeats are scrambled, i.e., theycontain a different amino acid sequence but retain the same amino acidcomposition. For example, a repeat may have a different amino acidsequence than SEQ ID NO: 8, but retain the same amino acid composition.

In some embodiments, the polypeptide with phase behavior is anelastin-like polypeptide. Elastin-like polypeptides (ELPs) arebiopolymers derived from tropoelastin. In some embodiments, theelastin-like polypeptides described herein are polymers comprising apentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly)_(n) (SEQID NO: 189). In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or500, including all values and ranges in between.

In some embodiments, the pentapeptide repeat is scrambled, for exampleit comprises a different amino acid sequence but maintains the sameamino acid composition. For example, an ELP may comprise a differentamino acid sequence than SEQ ID NO: 189, but maintains the same aminoacid composition, e.g. 40 % of the sequence is glycine, 20 % of thesequence is Xaa, 20 % of the sequence is proline, and 20 % of thesequence is valine.

In some embodiments, the ELP comprises one or more partial repeats. Insome embodiments, the length of a partial repeat is 1, 2, 3, or 4 aminoacids. In some embodiments, the ELP comprises one or more additionalamino acids at the N-terminus or C-terminus of the ELP that are not partof a repeat.

ELPs and RLPs undergo a phase transition in response to an environmentalfactor. ELPs and RLPs retain their ability to undergo a phase transitionwhen coupled to one or more polypeptides (such as one or more capturedomains), or expressed as a fusion protein with one or more otherpolypeptides (such as one or more capture domains). Polymers like ELPsand RLPs exhibit a transition temperature (Tt), also referred to as acloud point temperature (T_(c)). In some embodiments ELPs and RLPsundergo a reversible phase transition from a soluble to an insolublephase at the Tt. ELPs that transition from a soluble to an insolublephase with heating or an increase in salt concentration have a Ttreferred to as a lower critical solution temperature (LCST). RLPs thattransition from a soluble to an insoluble phase with cooling or adecrease in salt concentration have a Tt referred to as a lower criticalsolution temperature (UCST). In some embodiments, the phase transitionresults from a change in secondary structure of the ELP and/or RLP. Forexample, the phase transition of an ELP results from a change insecondary structure from a random coil (below the T_(t)) to a type IIβ-turn. In some embodiments, the change in secondary structure ischaracterized by a method selected from circular dichroismspectropolarimetry, small angle x-ray scattering, and cryo-electronmicroscopy, ultraviolet-visible spectrophotometry, static lightscattering, dynamic light scattering, nuclear magnetic resonancespectroscopy, solid-state nuclear magnetic resonance spectroscopy,infrared spectroscopy, Fourier transform infrared spectroscopy (FTIR),microscopy, and small angle neutron scattering. In some embodiments, thephase transition of an ELP does not result from a chance in secondarystructure.

In some embodiments, the RLPs and ELPs described herein have atransition temperature between about 0° C. and about 100° C. In someembodiments, the RLPs and ELPs described herein have a transitiontemperature between about 10° C. and about 50° C. In some embodimentsthe transition temperature is about 0° C., about 1° C., about 2° C.,about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13°C., about 14° C., about 15° C., about 16° C., about 17° C., about 18°C., about 19° C., about 20° C., about 21° C., about 22° C., about 23°C., about 24° C., about 25° C., about 26° C., about 27° C., about 28°C., about 29° C., about 30° C., about 31° C., about 32° C., about 33°C., about 34° C., about 35° C., about 36° C., about 37° C., about 38°C., about 39° C., about 40° C., about 41° C., about 42° C., about 43°C., about 44° C., about 45° C., about 46° C., about 47° C., about 48°C., about 49° C., about 50° C., about 51° C., about 52° C., about 53°C., about 54° C., about 55° C., about 56° C., about 57° C., about 58°C., about 59° C., about 60° C., about 61° C., about 62° C., about 63°C., about 64° C., about 65° C., about 66° C., about 67° C., about 68°C., about 69° C., about 70° C., about 71° C., about 72° C., about 73°C., about 74° C., about 75° C., about 76° C., about 77° C., about 78°C., about 79° C., about 80° C., about 81° C., about 82° C., about 83°C., about 84° C., about 85° C., about 86° C., about 87° C., about 88°C., about 89° C., about 90° C., about 91° C., about 92° C., about 93°C., about 94° C., about 95° C., about 96° C., about 97° C., about 98°C., about 99° C., or about 100° C. In some embodiments, the RLPsdescribed herein have a transition temperature from about 10° C. toabout 100° C.

In some embodiments, the Tt of the RLPs and ELPs described herein ismodulated by manipulating the primary structure e.g. amino acid sequenceof the RLP and ELP. In some embodiments, the hydrophobicity of the ELPor RLP is modulated. In some embodiments, the hydrophobicity of the ELPis modified by altering the identity of the guest residue Xaa. In someembodiments, the hydrophobicity of the ELP or RLP is increased resultingin a decreased Tt. In some embodiments, the hydrophobicity of the ELP orRLP is decreased resulting in an increased Tt. In some embodiments, thepolarity of the ELP or RLP is modulated. In some embodiments, thepolarity of the ELP is modulated by altering the identity of the guestresidue Xaa. In some embodiments, the polarity of the ELP or RLP isincreased resulting in an increased Tt. In some embodiments, thepolarity of the ELP or RLP is decreased resulting in a decreased Tt.

In some embodiments, the number of ELP pentapeptide repeats (n) ismodulated to alter the Tt. In some embodiments, n of the pentapeptiderepeat (Val-Pro-Gly-Xaa-Gly)_(n) (SEQ ID NO: 189) is an integer from 1to 500, inclusive of endpoints. In some embodiments, n is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, or 500, including all values and ranges in between.

In some embodiments, Xaa also referred to herein as “the guest residue”is any amino acid that does not eliminate the phase behavior of the ELP.In some embodiments, Xaa is any amino acid except proline. In someembodiments, Xaa is independently selected for each repeat. For example,a given ELP may contain the guest residues alanine, glycine, and valineat a ratio of 8:7:1. In some embodiments Xaa is selected from the groupconsisting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, praline, serine, threonine,tryptophan, tyrosine and valine. In some embodiments, Xaa is anon-classical amino acid selected from Table 2 and/or the groupconsisting of 2,4-diaminobutyric acid, α-amino-isobutyric acid,alloisoleucine, 4-aminobutyric acid, 2-amino butyric acid (Abu), γ-Abu,ε-Ahx, 6-amino hexanoic acid, 2-amino isobutyric acid (Aib), 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Cα-methyl amino acids, Na-methyl amino acids, and amino acid analogs ingeneral. In some embodiments, Xaa is the D-isomer of a natural ornon-classical amino acid.

In some embodiments, the Tt of the RLPs and ELPs described herein ismodulated by introducing one or more environmental factors to thecomposition containing the RLP and/or ELP. In some embodiments, the Ttof the ELPs and/or RLPs is modulated by adjusting the ionic strength ofsolvents. In some embodiments, the ionic strength of the solvent isadjusted by adding salt. In some embodiments, ELPs and/or RLPs containlower T_(t) in solvents containing anions categorized as kosmotropes.Anions that are kosmotropes are highly hydrated and influence the watershield on ELPs and/or RLPs. In some embodiments, the T_(t) of ELPsand/or RLPs can be adjusted through the addition of anions that arechaotropes. At low concentrations, the addition of chaotropes increasethe Tt of the ELP and/or RLP. At high concentrations, the addition of achaotrope decreases the T_(t) of the ELP and/or RLP. In someembodiments, the T_(t) of the ELP and/or RLP can be tuned by introducingone or more reagents that disrupts hydrogen bonds. Non-limiting examplesof reagents that disrupt hydrogen bonds include sodium dodecyl sulfate(SDS) and urea. In some embodiments, reagents that enhance hydrogen bondformation are utilized to modulate the Tt. In some embodiments, reagentsthat enhance hydrophobic interactions are utilized to modulate the Tt.Trifluoroethanol is a reagent which enhances both hydrophobicinteractions and hydrogen bond formation, causing a decrease in Tt.

In some embodiments, the ELP and/or RLP concentration can be adjusted tomodulate Tt. In some embodiments, a higher ELP and/or RLP concentrationresults in a reduced Tt. In some embodiments, a lower ELP and/or RLPconcentration results in an increased Tt.

In addition, modulation of pH, light, and ion concentrations also can beutilized to modulate T_(t).

In some embodiments, modulation of the number of (e.g. addition orremoval) charged amino acids (e.g. histidine, lysine, arginine, glutamicacid, aspartic acid, ornithine, or other non-natural charged aminoacids) and identity (e.g. positively or negatively charged) enablestuning of the Tt through pH modulation.

In some embodiments, the ELPs and/or RLPs described herein are blockcopolymers. A block copolymer comprises two or more sequence domains orblocks, in which two or more blocks contain different properties.Non-limiting examples of properties that can be tuned includehydrophilicity, hydrophobicity, polarity, and secondary structure. Insome embodiments, the block copolymer is an amphiphile, e.g. itcomprises at least one hydrophobic and at least one hydrophilic block.

In some embodiments, the ELPs and/or RLPs described herein assemble intovarious morphologies. Non-limiting examples of morphologies include aspherical aggregate, a micelle, a vesicle, a fibril, a nanofibril, ananotube, and a hydrogel. In some embodiments, the RLPs and/or ELPsdescribed herein assemble into various morphologies after the additionof an environmental factor. In some embodiments, the RLPs and/or ELPsdescribed herein change from one morphology to another morphology afterthe addition of an environmental factor. In some embodiments, the RLPsand/or ELPs described herein change from one morphology to anothermorphology after the addition of a biologic.

In some embodiments, addition of an environmental factor causes an RLPand/or ELP to undergo a phase transition. In some embodiments, at theRLP and/or ELP phase transition, the RLP and/or ELP converts from onemorphology to another morphology.

In some embodiments, a phase transition of an RLP and/or ELP causes theformation of dense, liquid, droplets.

In some embodiments, the polypeptide with phase behavior comprises anamino acid sequence selected from the group consisting of:

-   (a) (GRGDSPY)_(n) (SEQ ID NO: 180)-   (b) (GRGDSPH)_(n) (SEQ ID NO: 181)-   (c) (GRGDSPV)_(n) (SEQ ID NO: 182)-   (d) (GRGDSPYG)_(n) (SEQ ID NO: 183)-   (e) (RPLGYDS)_(n) (SEQ ID NO: 184)-   (f) (RPAGYDS)_(n) (SEQ ID NO: 185)-   (g) (GRGDSYP)_(n) (SEQ ID NO: 186)-   (h) (GRGDSPYQ)_(n) (SEQ ID NO: 187)-   (i) (GRGNSPYG)_(n) (SEQ ID NO: 188)-   (j) (GVGVP)n(SEQ ID NO: 190);-   (k) (GVGVPGLGVPGVGVPGLGVPGVGVP)_(m) (SEQ ID NO: 12);-   (l) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 13);-   (m) (GVGVPGWGVPGVGVPGWGVPGVGVP)_(m) (SEQ ID NO: 14);-   (n) (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)_(m) (SEQ ID NO:    15);-   (o) (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)_(m) (SEQ ID NO:    16); and-   (p) (GAGVPGVGVPGAGVPGVGVPGAGVP)_(m) (SEQ ID NO: 17);-   or a randomized, scrambled analog thereof; wherein:-   n is an integer in the range of 1-500, inclusive of endpoints; and-   m is an integer in the range of 4-25, inclusive of endpoints.

In some embodiments, the polypeptide with phase behavior comprises anamino acid sequence of (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 53)or (GVGVPGVGVPGLGVPGVGVPGVGVP)_(m) (SEQ ID NO: 55), wherein m is aninteger between 2 and 32, inclusive of endpoints. In some embodiments,the polypeptide with phase behavior comprises an amino acid sequence of(GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 193), wherein m is 8 or 16.In some embodiments, the polypeptide with phase behavior comprises anamino acid sequence of (GVGVPGAGVP)_(m) (SEQ ID NO: 54), wherein m is aninteger between 5 and 80, inclusive of endpoints. In some embodiments,the polypeptide with phase behavior comprises an amino acid sequence of(GXGVP)_(m) (SEQ ID NO: 56), wherein m is an integer between 10 and 160,inclusive of endpoints, and wherein X for each repeat is independentlyselected from the group consisting of glycine, alanine, valine,isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine,arginine, aspartic acid, glutamic acid, and serine.

In some embodiments, the polypeptide with phase behavior comprises anamino acid sequence selected from

-   (a) (GVGVP)_(m) (SEQ ID NO: 52);-   (b) (ZZPXXXXGZ)_(m) (SEQ ID NO: 57);-   (c) (ZZPXGZ)_(m) (SEQ ID NO: 58);-   (d) (ZZPXXGZ)_(m) (SEQ ID NO: 59); and-   (e) (ZZPXXXGZ)_(m) (SEQ ID NO: 60),

wherein m is an integer between 10 and 160, inclusive of endpoints,wherein X if present is any amino acid except proline or glycine, andwherein Z if present is any amino acid.

In some embodiments, the polypeptide with phase behavior comprises anamino acid sequence of (GVGVP)_(m) (SEQ ID NO: 192), wherein m is 20,40, or 80. In some embodiments, the polypeptide with phase behaviorcomprises an amino acid sequence of (GRGDXPZX)_(m) (SEQ ID NO: 61) or(XZPXDGRG)_(m) (SEQ ID NO: 51), wherein X is glutamine or serine, Z istyrosine or valine, and m is an integer between 10 and 160, inclusive ofendpoints.

In some embodiments, the polypeptide with phase behavior comprises afirst set of repeat sequences and a second set of repeat sequences. Thefirst set of repeat sequences and the second set of repeat sequences mayeach individually comprise sequences that are repeated one or moretimes. In some embodiments, the first set of repeat sequences any/or thesecond set of repeat sequences comprises a repeating sequence comprisingany one of SEQ ID NOs: 1-17 and 51-61. In some embodiments, thepolypeptide with phase behavior comprises a first set of repeatsequences and a second set of repeat sequences, wherein the first set ofrepeat sequences comprises the amino acid sequence of (GRGDXPZX)₄₀ (SEQID NO: 149) and the second set of repeat sequences comprises the aminoacid sequence (GVGVP)₈₀ (SEQ ID NO: 150), wherein X is glutamine and Zis tyrosine. In some embodiments, polypeptide with phase behaviorcomprising a first set of repeat sequences and a second set of repeatsequences comprises the sequence of SEQ ID NO: 151 In some embodiments,the polypeptide with phase behavior comprises at least one, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, or at least ten differentsets of repeat sequences. In some embodiments, each set of repeatsequences within the polypeptide with phase behavior comprises sequencesthat repeat from about 5 to about 400 times, for example, about 5, about10, about 15, about 20, about 25, about 30, about 35, about 40, about45, about 50, about 55, about 60, about 65, about 70, about 75, about80, about 85, about 90, about 95, about 100, about 105, about 110, about115, about 120, about 125, about 130, about 135, about 140, about 145,about 150, about 155, about 160, about 165, about 170, about 175, about180, about 185, about 190, about 195, about 200, about 205, about 210,about 215, about 220, about 225, about 230, about 235, about 240, about245, about 250, about 255, about 260, about 265, about 270, about 275,about 280, about 285, about 290, about 295, about 300, about 305, about310, about 315, about 320, about 325, about 330, about 335, about 340,about 345, about 350, about 355, about 360, about 365, about 370, about375, about 380, about 385, about 390, about 395, or about 400 times.

In some embodiments, the polypeptide with phase behavior comprising anamino acid sequence selected from any one of SEQ ID NOs: 1-17 and 51-61also comprises up to 10 additional N-terminal and/or C-terminal aminoacids. In some embodiments, the polypeptide with phase behaviorcomprising an amino acid sequence of any one of SEQ ID NOs: 1-17 and51-61 also comprises an additional N-terminal methionine. In someembodiments, the polypeptide with phase behavior comprising an aminoacid sequence of any one of SEQ ID NOs: 1-17 and 51-61 also comprises anadditional C-terminal glycine.

In some embodiments, the polypeptide with phase behavior has the sameamino acid composition of an ELP and/or RLP but does not containrepeats. In some embodiments, the polypeptide with phase behaviorcomprises an amino acid sequence that is 80 %, about 85 %, about 90 %,about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or about 100% identical to an ELP and/or RLP. In some embodiments, the polypeptidewith phase behavior comprises an amino acid composition that is 80 %,about 85 %, about 90 %, about 95 %, about 96 %, about 97 %, about 98 %,about 99 %, or about 100 % identical to an ELP and/or RLP. In someembodiments, the polypeptide with phase behavior comprises a compositionof hydrophobic amino acids that is 80 %, about 85 %, about 90 %, about95 %, about 96 %, about 97 %, about 98 %, about 99 %, or about 100 %identical to an ELP and/or RLP.

In some embodiments, the polypeptide with phase behavior comprises anon-repetitive unstructured polypeptide. In some embodiments, thenon-repetitive unstructured polypeptide has an amino acid sequence thatcontains at least 50 amino acids. In some embodiments, thenon-repetitive unstructured polypeptide has an amino acid sequence thatcontains at least 50, at least 60, at least 70, at least 80, at least90, or at least 100 amino acids. In some embodiments, the sequence ofthe non-repetitive unstructured polypeptide is at least about 10 %proline (e.g. at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, or 80%) and at least 20 % glycine (e.g. at least 20 %, at least 30 %, atleast 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %,or at least 90 %). In some embodiments, the non-repetitive unstructuredpolypeptide has a sequence that contains at least about 40 % of aminoacids selected from the group consisting of valine, alanine, leucine,lysine, threonine, isoleucine, tyrosine, serine, and phenylalanine.

In some embodiments, the non-repeated unstructured polypeptide comprisesa sequence that does not contain three contiguous identical amino acids,wherein any 5-10 amino acid subsequence does not occur more than once inthe non-repeated unstructured polypeptide, and wherein when thenon-repeated unstructured polypeptide comprises a subsequence startingand ending with proline, and wherein the subsequence further comprisesat least one glycine.

In some embodiments, the ELPs and/or RLPs described herein are expressedas a component of a fusion protein. In some embodiments, the fusionprotein is expressed in bacteria or mammalian cells. In someembodiments, the fusion protein is expressed in Escherichia coli. Insome embodiments, the fusion protein is expressed in insect cells. Insome embodiments, the sequence of the non-repetitive unstructuredpolypeptide is at least about 10 % proline (e.g. at least 10 %, 20 %, 30%, 40 %) and at least 20 % glycine (e.g. at least 20 %, 30 %, 40 %, or50 %), and at least 40 % (e.g. at least 40 %, 50 %, 60 %, or 70 %) ofamino acids selected from the group consisting of valine, alanine,leucine, lysine, threonine, isoleucine, tyrosine, serine, andphenylalanine.

In some embodiments, the non-repetitive unstructured polypeptide doesnot contain three contiguous identical amino acids. In some embodiments,the non-repetitive unstructured polypeptide comprises a subsequence(e.g. a fragment of the non-repetitive unstructured polypeptide) whichonly occurs once in the non-repetitive unstructured polypeptidesequence. In some embodiments, the non-repetitive unstructuredpolypeptide comprises a subsequence that starts and ends with proline.In some embodiments, the non-repetitive unstructured polypeptidecomprises a subsequence that contains at least one glycine.

In some embodiments, the polypeptide with phase behavior comprises asignal peptide.

In some embodiments, the polypeptide with phase behavior comprises anamino acid sequence of (GVGVPGLGVPGVGVPGLGVPGVGVP)_(m) (SEQ ID NO: 191),wherein m is 16. In some embodiments, the polypeptide with phasebehavior comprises an amino acid sequence of SEQ ID NO: 164. In someembodiments, the polypeptide with phase behavior comprises an amino acidsequence of SEQ ID NO: 165.

Linkers Between Polypeptides With Phase Behavior and Capture Domains

In some embodiments, the capture domain is coupled to the polypeptidewith phase behavior via a linker. In some embodiments, any linker thatdoes not interfere with the function of the purification matrix may beutilized.

In some embodiments, the linker connects the capture domain to thepolypeptide with phase behavior. In some embodiments, the linker enablescooperative interactions between the polypeptide with phase behavior andthe capture domain. In some embodiments, the linker is a peptide. Insome embodiments, the linker preserves the phase behavior of thepolypeptide with phase behavior. In some embodiments, the linkerpreserves the Tt of the polypeptide with phase behavior. In someembodiments, the linker preserves the structure of the capture domain.In some embodiments, the linker comprises between 1 and 50 amino acids.In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 amino acids.

In some embodiments, the stiffness of the linker is increased by theinclusion of proline in the linker amino acid sequence.

In some embodiments, the flexibility of a linker is increased by theinclusion of small polar amino acids, including threonine, serine, andglycine.

In some embodiments, the linker may adopt various secondary structures,including but not limited to α-helices, β-strands, and random coils. Insome embodiments, the linker adopts an α-helix and comprises an aminoacid repeat of (EAAAK)_(n) (SEQ ID NO: 18) where n is a repeat number,i.e.., an integer in the range of 1 to 20, inclusive of endpoints.

In some embodiments, the linker is comprised of (G₄S)_(n) (SEQ ID NO:19) where n can be an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30). In embodiments, the polypeptide linker has arepeat of (SGGG)n (SEQ ID NO: 20), wherein n is an integer from 1 to 50(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20). In embodiments, the polypeptide linker has a repeat of (GGGS)_(n)(SEQ ID NO: 21), wherein n is an integer from 1 to 20 (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In some embodiments, the linker has an amino acid sequence ofKESGSVSSEQLAQFRSLD (SEQ ID NO: 22). In some embodiments, the linker hasan amino acid sequence of EGKSSGSGSESKST (SEQ ID NO: 23). In someembodiments, the linker only contains glycine.

In some embodiments, the peptide linker comprises a protease cleavagesite. In some embodiments, the protease cleavage site is a furincleavage site.

In some aspects, the polypeptide linker is a poly-(Gly)_(n) linker,wherein n is an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30(SEQ ID NO: 50). In other embodiments, the linker is selected from thegroup consisting of: dipeptides, tripeptides, and quadripeptides. Inembodiments, the linker is a dipeptide selected from the groupconsisting of alanine-serine (AS), leucine-glutamic acid (LE), andserine-arginine (SR).

In some embodiments, the linker is selected from GKSSGSGSESKS (SEQ IDNO: 152), GSTSGSGKSSEGKG (SEQ ID NO: 153), GSTSGSGKSSEGSGSTKG (SEQ IDNO: 154), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 155), EGKSSGSGSESKEF (SEQ IDNO: 156), SRSSG (SEQ ID NO: 157), and SGSSC (SEQ ID NO: 158).

In some embodiments, the linker is a self-cleaving peptide. In someembodiments, the self-cleaving peptide is a 2A peptide. 2A peptides area class of 18-22 amino acid long peptides that induce ribosomal skippingduring translation of a protein in a cell. In some embodiments, the 2Apeptide is a T2A peptide having an amino acid sequence ofEGRGSLLTCGDVEENPGP (SEQ ID NO: 159), a P2A peptide having an amino acidsequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 160), an E2A peptide havingan amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 161), or anF2A peptide having an amino acid sequence of VKQTLNFDLLKLAGDVESNPGP (SEQID NO: 162). In some embodiments, the 2A peptide has at least 80 %, atleast 85 %, at least 90 %, at least 95 %, or at least 98 % identity toany one of SEQ ID NOs. 159-162. In some embodiments, the 2A peptidefurther comprises GSG (SEQ ID NO: 163) on its N-terminus.

In some embodiments, the linker is a chemical linker. In someembodiments, the chemical linker is selected from the group consistingof a carbohydrate linker, a lipid linker, a fatty acid linker, and apolyether linker.

In some embodiments, the linker is a direct covalent linkage between anamino acid residue of the polypeptide with an amino acid residue of thepolypeptide with phase behavior and a capture domain. In someembodiments, a fusion protein comprises the polypeptide with phasebehavior and a capture domain. In some embodiments, the fusion proteinfurther comprises one or more linkers as described herein. In someembodiments, a fusion protein comprises, from N-terminus to C-terminus,a polypeptide with phase behavior, a linker, and a capture domain. Insome embodiments, a fusion protein comprises, from N-terminus toC-terminus, a capture domain, a linker, and a polypeptide with phasebehavior.

ENVIRONMENTAL FACTORS

In some embodiments, one or more environmental factors are applied tocause a change of a complex comprising the protein-based purificationmatrix and biologic, contaminant, and/or other molecule. In someembodiments, the one or more environmental factors cause the size of acomplex comprising the protein-based purification matrix and biologic,contaminant, and/or other molecule to increase. In some embodiments, theone or more environmental factors cause the polypeptide with phasebehavior to aggregate. In some embodiments, the one or moreenvironmental factors causes separation of the protein-basedpurification matrix from the biologic, contaminant, and/or smallmolecule. In some embodiments, the one or more environmental factorsenables the biologic, contaminant, and/or other molecule to retain itsnative structure, function, and activity.

In some embodiments, the environmental factor is a change intemperature. In some embodiments, the temperature is increased about0.5° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5°C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C.,about 11° C., about 12° C., about 13° C., about 14° C., about 15° C.,about 16° C., about 17° C., about 18° C., about 19° C., about 20° C.,about 21° C., about 22° C., about 23° C., about 24° C., about 25° C.,about 26° C., about 27° C., about 28° C., about 29° C., about 30° C.,about 31° C., about 32° C., about 33° C., about 34° C., about 35° C.,about 36° C., about 37° C., about 38° C., about 39° C., or about 40° C.In some embodiments, the temperature is decreased about 0.5° C., about1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C.,about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about12° C., about 13° C., about 14° C., about 15° C., about 16° C., about17° C., about 18° C., about 19° C., about 20° C., about 21° C., about22° C., about 23° C., about 24° C., about 25° C., about 26° C., about27° C., about 28° C., about 29° C., about 30° C., about 31° C., about32° C., about 33° C., about 34° C., about 35° C., about 36° C., about37° C., about 38° C., about 39° C., or about 40° C.

In some embodiments, the environmental factor is a change in pH. In someembodiments, the pH is increased by about 0.1, about 0.2, about 0.3,about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9,about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2,about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5,about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0 units. In someembodiments, the pH is decreased by about 0.1, about 0.2, about 0.3,about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9,about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2,about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5,about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0 units.

In some embodiments, the environmental factor is change in ionicstrength. In some embodiments, the change in ionic strength is broughtabout by increasing the concentration of salt. In some embodiments, thechange in ionic strength is brought about by decreasing theconcentration of salt. Non-limiting examples of salts include sodiumchloride, potassium chloride, ammonium chloride, sodium acetate, sodiumcitrate, copper sulfate, sodium iodide, ammonium sulfate, and sodiumsulfate. In some embodiments, dialysis is used to change theconcentration of salt in the composition containing the protein-basedpurification matrix and biologic, contaminant, and/or other molecule.

In some embodiments, the environmental factor is the addition of acofactor. Non-limiting examples of cofactors include calcium, magnesium,cobalt, copper, zinc, iron, manganese, selenium, molybdenum, potassium,coenzyme A (CoA), a nucleoside triphosphate, and a vitamin. In someembodiments, the cofactor is calcium. In some embodiments, thenucleoside triphosphate is adenosine triphosphate, uridine triphosphate,guanosine triphosphate, cytidine triphosphate, or thymidinetriphosphate. In some embodiments, the vitamin is a fat-soluble. In someembodiments, the vitamin is water-soluble. Non-limiting examples ofvitamins include vitamin A, vitamin B1 (thiamine), vitamin B2(riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5(pantothenic acid ), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine,or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folicacid), vitamin B12, vitamin C, vitamin D, Vitamin E, vitamin K, K1, andK2, folic acid, and biotin.

In some embodiments, the environmental factor is a change in theconcentration of the protein-based purification matrix. In someembodiments, the environmental factor is a change in the concentrationof the biologic, contaminant, and/or other molecule.

In some embodiments, the environmental factor is a change in pressure ofthe composition containing the protein-based purification matrix andbiologic, contaminant, and/or other molecule. In some embodiments, achange in pressure can be effected by increasing or decreasing thevolume of the composition.

In some embodiments, the environmental factor is the addition of one ormore surfactants. In some embodiments, the one or more surfactants areselected from free fatty acid salts, soaps, fatty acid sulfonates, suchas sodium lauryl sulfate, ethoxylated compounds, such as ethoxylatedpropylene glycol, lecithin, polygluconates, quaternary ammonium salts,lignin sulfonates, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), sugars, including sucroseand glucose, Triton X-100, and NP-40. In some embodiments, thesurfactant is anionic, nonionic, or amphoteric.

In some embodiments, the environmental factor is the addition of one ormore molecular crowding agents. Non-limiting examples of molecularcrowding agents include polyethylene glycol, dextran, and ficoll. PEGSmay include PEG400, PEG1450, PEG3000, PEG8000, and PEG10000.

In some embodiments, the environmental factor is the addition of one ormore oxidizing agents. Non-limiting examples of oxidizing agents includehydrogen peroxide, hydrophilically or hydrophobically activated hydrogenperoxide, preformed peracids, monopersulfate or hypochlorite.

In some embodiments, the environmental factor is the addition of one ormore reducing agents. In some embodiments, the one or more reducingagents is selected from the group consisting of dithiothreitol (DTT),2-mercaptoethanol (BME), Tris (2-carboxyethyl) phosphine (TCEP),hydrazine, boron hydrides, amine boranes, lower alkyl substituted amineboranes, triethanolamine, and N,N,N′,N′-tetramethylethylenediamine(TEMED).

In some embodiments, the environmental factor is the addition of one ormore denaturing agents. Non-limiting examples of denaturing agentsinclude urea, guanidine hydrochloride, guanidine, sodium salicylate,dimethyl sulfoxide, and propylene glycol.

In some embodiments, the environmental factor is the addition of one ormore enzymes. Non-limiting examples of enzymes include proteases,kinases, phosphatases, synthetases, transferases, nucleases such asrestriction endonucleases, lyases, isomerases, dehydrogenases,decarboxylases, and lipases.

In some embodiments, the environmental factor is the application ofelectromagnetic waves. In some embodiments, the environmental factor isthe application of light. In some embodiments, the electromagnetic waveshave a wavelength between about 0.0001 nm and about 100 m. In someembodiments, the electromagnetic waves are selected from the groupconsisting of gamma rays, x-rays, ultraviolet, visible, infrared, andradio waves. In some embodiments, the electromagnetic waves are gammarays. In some embodiments, the gamma rays have a wavelength betweenabout 0.0001 nm and about 0.01 nm, e.g. 0.0001 nm, 0.0005 nm, 0.001 nm,0.002 nm, 0.003 nm, 0.004 nm, 0.005 nm, 0.006 nm, 0.007 nm, 0.008 nm,0.009 nm, and 0.01 nm. In some embodiments, the x-rays have a wavelengthbetween about 0.01 nm and 10 nm, e.g. about 0.01 nm, 0.02 nm, 0.03 nm,0.04 nm, 0.05 nm, 0.06 nm, 0.07 nm, 0.08 nm, 0.09 nm, 0.10 nm, 0.2 nm,0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm, 3nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or about 10 nm. In someembodiments, the ultraviolet radiation has a wavelength between about 10nm about 400 nm, e.g. about 10 nm, about 20 nm, about 30 nm, about 40nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm,about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 280 nm,about 300 nm, about 350 nm, or about 400 nm. In some embodiments, thevisible waves have a wavelength of between about 400 nm and about 800nm, e.g. about 400 nm, about 450 nm, about 500 nm, about 550 nm, about600 nm, about 650 nm, about 700 nm, about 750 nm, or about 800 nm. Insome embodiments, the infrared radiation has a wavelength of betweenabout 800 nm and about 0.1 cm, e.g. about 800 nm, about 1 µm, about 2µm, about 3 µm, about 4 µm, about 5 µm, about 6 µm, about 7 µm, about 8µm, about 9 µm, about 10 µm, about 20 µm, about 30 µm, about 40 µm,about 50 µm, about 60 µm, about 70 µm, about 80 µm, about 90 µm, about100 µm, about 200 µm, about 300 µm, about 400 µm, about 500 µm, about600 µm, about 700 µm, about 800 µm, about 900 µm, or about 0.1 cm. Insome embodiments, the radio waves has a wavelength of between about 0.1cm and 100 m, e.g. about 0.1 cm, about 1 cm, about 10 cm, about 100 cm,about 1000 cm, about 2000 cm, about 3000 cm, about 4000 cm, about 5000cm, about 6000 cm, about 7000 cm, about 8000 cm, about 9000 cm, or about100 m.

In some embodiments, the environmental factor is the application ofacoustic waves. In some embodiments, the acoustic waves have a frequencybetween about 1 Hz and 2000 kHz. In some embodiments, the acoustic waveshave a frequency of about 1 Hz, about 5 Hz, about 10 Hz, about 20 Hz,about 30 Hz, about 40 Hz, about 50 Hz, about 60 Hz, about 70 Hz, about80 Hz, about 90 Hz, about 100 Hz, about 200 Hz, about 300 Hz, about 400Hz, about 500 Hz, about 600 Hz, about 700 Hz, about 800 Hz, about 900Hz, about 1 kHz, about 100 kHz, about 200 kHz, about 300 kHz, about 400kHz, about 500 kHz, about 600 kHz, about 700 kHz, about 800 kHz, about900 kHz, about 1000 kHz, about 1100 kHz, about 1200 kHz, about 1300 kHz,about 1400 kHz, about 1500 kHz, about 1600 kHz, about 1700 kHz, about1800 kHz, about 1900 kHz, or about 2000 kHz.

EXAMPLES Example 1. Development of Protein-Based Purification MatricesThat Exhibit Phase Behavior and Bind to a Biologic

A protein-based purification matrix is generated and characterized.Initially, an expression vector comprising a sequence encoding a capturedomain that binds to the Fc region of an antibody and a sequenceencoding a polypeptide with phase behavior (e.g., an ELP) is generated.A fusion protein comprising the capture domain and polypeptide withphase behavior is expressed in Escherichia coli according to standardprotocols. Isothermal titration calorimetry is utilized to characterizedthe affinity of the fusion protein for pure human immunoglobulin G(IgG). The transition temperature of the fusion protein is determinedusing UV-Vis spectrophotometry.

Example 2. Utilization of Protein-Based Purification Matrices ThatExhibit Phase Behavior and Bind to a Biologic to Purify Protein

The protein-based purification matrix of Example 1 is utilized to purifyan antibody such as trastuzumab (Herceptin®). Various molar ratios ofprotein-based purification matrix to biologic (e.g. trastuzumab) aretested to determine an optimal ratio for purification (i.e., forcomplete capture of the biologic). An environmental factor (e.g.,addition of salt such as sodium chloride or ammonium sulfate) is appliedto the composition containing the protein-based purification matrix andthe biologic to cause the protein-based purification matrix increase insize.

Both tangential flow filtration and centrifugation are utilized toseparate the biologic from the protein-based purification matrix.Although both centrifugation and tangential flow filtration enableseparation of the biologic from the protein-based purification matrix,tangential flow filtration is preferred because it enables the rapidpurification of thousands of liters of sample volume without therequirement for specialized centrifuges.

TFF is performed using standard conditions - for example, a 1.5 bartransmembrane pressure and a 960 L/m²/h cross flow rate. Standard TFFmembranes are used, such as a 0.1 µm hydrophilized poly(vinylidenedifluoride) (PVDF) or a 0.2 µm polyethersulfone membrane.

The pH is adjusted (for example to an acidic pH such as 2-4.5, or pH 3)to separate (i.e. elute) the protein-based purification matrix from thebiologic. The purity of the biologic is characterized by size exclusionchromatography. The protein-based purification matrix is then reused foran additional round of purification.

Example 3. Utilization of Protein-Based Purification Matrices ThatExhibit Phase Behavior and Bind to a Biologic to Purify Cell

A protein-based purification matrix is utilized to purify T cells. Thecapture domain of the protein-based purification matrix specificallybinds to T cells, for example, by recognizing a T-cell specific surfacemarker such as CD3, CD4, or CD8. Various molar ratios of protein-basedpurification matrix to T cell are tested to determine an optimal ratiofor purification. Various environmental factors are tested, such asdifferent salts and salt concentrations, to determine the mostefficacious way of isolating T cells.

Tangential flow filtration and/or centrifugation and/or continuouscentrifugation are utilized to separate the T cell from theprotein-based purification matrix.

Example 4. Purification of AAV9 Using a Purification Matrix andTangential Flow Filtration (TFF)

HEK293 cells producing a recombinant AAV9 vector packaging a tdTomatotransgene were grown in suspension and harvested by centrifugation. 200mL of the supernatant were treated with 10 U/mL benzonase and 0.01%pluronic acid and sterile filtered through a 0.2-micron bottle-topfilter. This starting material (SM) was then mixed with 1 µMpurification matrix having an amino acid sequence of SEQ ID NO: 172 and0.6 M NaCl salt (i.e., the first environmental factor) to form anAAV-purification matrix complex.

This SM was processed in continuous fed-batch mode using a Repligen KR2itangential-flow filtration (TFF) unit. The TFF was setup with a 20 mLretentate vessel, prepared with 50 µM purification matrix and 0.6 MNaCl, as well as a 13 cm² hollow fiber filter with 0.2-micron pores. Aconcentration-diafilter (CD) mode (10x concentration factor (CF), 6xdiavolumes (DV) was run in permeate control with the SM and permeatepump set to equal flow rates. Once the entire 200 mL SM feed wasprocessed, the retained material was rinsed with 6 DV of wash buffer (20mM Tris, 0.5 M NaCl). The AAV9 material, now substantially free ofcontaminants, was then resolubilized on ice and mixed with an equalvolume of 2x Elution buffer (i.e., a second environmental factor) withthe permeate valve closed. The elution buffer contained 100 mM glycineat pH 3 and 0.6 M NaCl. After 20 minutes, the recirculating sample waswarmed back to room temperature and phase separated with NaCl. Thepermeate valve was opened and pure AAV9 was collected in a second CDmode (2x CF, 4x DV) with elution buffer diavolumes (100 mM glycine, pH3, 0.6 M NaCl). The flux and transmembrane pressure (TMP) were trackedthroughout the run (FIG. 1 ), demonstrating a stable, efficient, andscalable process. Permeate and retentate samples were collectedthroughout the run for analysis of AAV loss by anti-AAV Western Blot(FIG. 2 ), as well as purity by host cell protein (HCP) quantificationusing a HEK HCP ELISA (Cygnus®) and dsDNA quantification using aQuant-iT™ picogreen assay® (Thermo Fisher®). The process enabled removalof HCPs by 2-3 log (FIG. 3 ) and of double stranded DNA (dsDNA) by >3log (FIG. 4 ).

Example 5. Effect of Titer, Clarification, and Nuclease Treatment on AAVPurification

The ability of purification matrix having an amino acid sequence of SEQID NO: 172 to capture AAV8 particles from cell lysate or the media ofsuspension cultures of HEK293 cells producing AAV8 particles (referredto in FIG. 10 as “supernatant”) was tested.

A cell lysate was produced by resuspending pelleted HEK293 cellsproducing AAV8 particles in 0.5 % Triton-X-100. The cell lysate andmedia evaluated contained titers of AAV particles that ranged from 1 ×10⁸ to 1 × 10⁷ viral particles per microliter (vp/µL). The ability ofpurification matrix to capture AAV8 particles from cell lysate treatedwith nuclease was also evaluated. Cell lysates that were treated withnuclease were incubated for 1 hr at 34° C. with 50 U/mL benzonase(Millipore^(®)).

The ability of purification matrix to capture AAV8 particles fromclarified cell lysate or media was also evaluated. Cell lysates and/ormedia were clarified by centrifugation at 13,200 rpm for 10 minutes. Thesupernatant was used for subsequent isolation of the AAV8 particles for10 min at 13,200 rpm.

AAV8 particles were isolated from each sample by mixing the sample with10 µM purification matrix and 0.6 M NaCl (i.e., a first environmentalfactor) and centrifuging the sample at 13,200 rpm for 10 minutes.

The pellets containing AAV-purification matrix complex were resuspendedon ice in an elution buffer (i.e., second environmental factor)comprising 100 mM glycine at pH 3, warmed to room temperature,transitioned with 0.6 M salt, and then centrifuged a second time. Theamount of eluted AAV8 was compared to that in the starting material,(i.e the media or cell lysate comprising AAV8 particles) using invertedterminal repeat (ITR) quantitative polymerase chain reaction (qPCR).This technique quantitates the number of AAV particles by measuring thenumber of ITRs using PCR. The AAV Capture Efficiency for each sample wascalculated using the following equation: 100 × (# of AAV8 particlescaptured by the purification matrix / # of AAV8 particles in thecomposition before purification).

The purification matrix robustly captured >98% AAV particles regardlessof titer (110⁷ to 4×10¹⁰), clarification, nuclease treatment, or lysateversus media as the starting material (FIG. 5 ). Evaluation of lysatesamples with and without nuclease treatment showed that nucleasetreatment did not impact final eluted AAV8 particle purity when comparedby silver stain SDS-PAGE (FIG. 6 ) and Quant-iT™ picogreen assay fordsDNA (FIG. 7 ).

Example 6. Effect of Centrifugation Speed on AAV8 Capture

Harvested media from AAV8 HEK293 suspension cell culture was mixed with10 µM of Purification Matrix having an amino acid sequence of SEQ ID NO:172 and 0.6 M NaCl to form an AAV-purification matrix complex. Thesamples were centrifuged for 10 min at relative centrifugal forces (RCF)ranging from 100 to 16,000. For each RCF, uncaptured AAV8 remaining inthe supernatant was quantified using ITR qPCR and calculated as apercentage of the amount measured in the starting harvest material. TheAAV Capture Efficiency was measured as 100% less the percentageremaining uncaptured in the supernatant and results showed that speedsof 500 RCF or higher yielded highly efficient AAV8 capture (FIG. 8 ).

Example 7. Stabilization of AAV2 by Purification Matrix

Adherent HEK293 cells were transfected according to a standard tripletransfection method, and used to produce recombinant AAV2 particlescarrying a luciferase transgene. The cells were cultured for 6 days, inthe presence or absence of purification matrix having an amino acidsequence of SEQ ID NO: 172. Cells cultured in the presence of 10 µMpurification matrix were compared to control cells, with each group,treatment or control, in triplicate. The culture media was collected onday 4 and replaced with an equal volume of media with or without thepurification matrix additive. On day 6, the media was collected againand the cells were rinsed with PBS and harvested by scraping. The totalvector genomes collected in all fractions were quantified for comparisonusing qPCR using primers against ITR2. Inclusion of the purificationmatrix in the culture media increased vector genome (vg) titers by atleast 8% (FIG. 9 ).

Example 8. Stabilization of AAV8 by Purification Matrix

Media was harvested from HEK293 cells grown in suspension, wherein thecells were producing GFP-AAV8. The media was aliquoted and stored at-20° C. either with or without the addition of 100 µM of purificationmatrix having an amino acid sequence of SEQ ID NO: 172. As anaccelerated stability study, the samples were subjected to freeze-thawcycles (-20° C. to room temperature) and then assayed using an AAV8ELISA (Progen), which quantifies total intact AAV particles. Thequantified particles for the control and purification matrix-treatedsamples were normalized to the starting materials with no freeze-thaws.This data indicates that the purification matrix may enhance theresistance of AAV particles to freeze-thaw-mediated degradation andaggregation (FIG. 10 ).

Example 9. Capture of AAV8 Particles With a Purification Matrix

Media was harvested from HEK293 suspension cells producing AAV8particles carrying a luciferase (luc) transgene. The media was contactedwith 0 µM, 0.1 µM, 0.5 µM, 1 µM, 10 µM, and 50 µM purification matrixhaving an amino acid sequence of SEQ ID NO: 172, resulting in theformation of complexes between AAV8 particles and purification matrix. Afirst environmental factor (i.e. 0.6 M NaCl) was applied to increase thesize of the complexes. Subsequently, the media containing the complexeswas centrifuged at 13,200 revolutions per minute (rpm) for 10 minutes(min). This protocol allowed for separation of the complexes fromimpurities on the basis of size. Inverted terminal repeat (ITR)quantitative polymerase chain reaction (qPCR) was utilized to evaluatethe amount of AAV particles captured from the media using thepurification matrix compared to the amount of AAV particles in thestarting material. This technique quantitates the number of AAVparticles by measuring the number of ITRs using PCR. The CaptureEfficiency of the purification matrix at each concentration wascalculated using the following equation: 100 x (# of AAV8 particlescaptured by the purification matrix / # of AAV8 particles in thecomposition before purification). FIG. 11 shows that purification matrixconcentrations at or above 10 µM are sufficient for robust, >98%, viruscapture.

Example 10. Capture of Ad5 Particles With a Purification Matrix

Media was harvested from HEK293 cells grown in suspension, wherein thecells were producing adenovirus type 5 (Ad5) particles carrying a greenfluorescent protein (GFP) transgene. The media was contacted with 0 µM,0.1 µM, 0.5 µM, 1 µM, 10 µM, and 50 µM purification matrix having anamino acid sequence of SEQ ID NO: 173, resulting in the formation ofcomplexes between Ad5 particles and purification matrix. A firstenvironmental factor (i.e. 0.6 M NaCl) was applied to increase the sizeof the complexes. Subsequently, the media containing the complexes wascentrifuged at 13,200 revolutions per minute (rpm) for 10 minutes (min).This protocol allowed for separation of the complexes from impurities onthe basis of size.

Flow cytometry was utilized to evaluate the amount of Ad5 particlescaptured from the media using the purification matrix compared to theamount of Ad5 particles in the starting material. The Capture Efficiencyof the purification matrix at each concentration was calculated usingthe following equation: 100 x (# of Ad5 particles captured by thepurification matrix / # of Ad5 particles in the composition beforepurification). FIG. 12 shows that purification matrix concentrations ator above 10 µM are sufficient for robust, >98%, virus capture.

Example 11. Capture of Lentivirus Particles With a Purification Matrix

Lentivirus particles carrying a green fluorescent protein (GFP)transgene were mixed with 10 µM of purification matrix or PBS (negativecontrol). Mixture of lentivirus particles with purification matrixhaving an amino acid sequence of SEQ ID NO: 174 leads to the formationof complexes between lentivirus particles and purification matrix. Afirst environmental factor (i.e. 0.5 M NaCl) was applied to increase thesize of the complexes. The composition containing lentivirus particlesand purification matrices, and the composition containing lentivirusparticles and PBS were centrifuged at 13,200 revolutions per minute(rpm) for 10 minutes (min). The supernatants of each composition, whichcontained uncaptured lentivirus particles, were added to the media ofadherent HEK293 cells for 48 hours. Subsequently, the cells werefluorescently imaged. HEK293 cells incubated with supernatant from thecomposition comprising lentivirus particles and PBS exhibited greaterfluorescence than HEK293 cells incubated with supernatant from thecomposition comprising lentivirus particles and purification matrix.

Thus, more lentivirus particles were contained in the supernatant of thecomposition containing lentivirus particles and PBS than the compositioncontaining lentivirus particles and purification matrix. This shows thatthe purification matrix captured lentivirus particles (FIG. 13 ).

This experiment is repeated with a purification matrix having an aminoacid sequence of SEQ ID NO: 175.

Example 12. Capture of Human Serum Albumin (HSA) With a PurificationMatrix

Human serum albumin (HSA) was contacted with a purification matrixhaving an amino acid sequence of SEQ ID NO: 176 for a period of time toallow complex formation. A first environmental factor was applied to thecomplexes (i.e. about 0.6 M NaCl) to increase the size of the complexes.

The composition comprising the HAS, purification matrix, and 0.5 M NaClwas applied to two filters, a filter with a 0.2 µm pore size and afilter with a 300 kilodalton (kDa) molecular weight cutoff. The filtratewas applied to a sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) gel, which was subsequently stained with aCoomassie dye.

The SDS-PAGE gel shows the presence of a band for HSA (at 66.5 kDa) inthe filtrate from the 0.2 µm pore size filter and the absence of theband for HSA in the filtrate from the 300 kDa filter. The 300 kDa filterretained > 90 % of the HSA. Thus, capturing HSA with a purificationmatrix, and increasing the size of the complexes with an environmentalfactor increases the size of HSA such that 90 % of the HSA is retainedby the filter (FIG. 14 ).

Example 13. Infectivity of Ad5 Particles in the Presence of PurificationMatrix

Media was harvested from HEK293 cells producing adenovirus type 5 (Ad5)particles carrying a green fluorescent protein (GFP) transgene. Themedia containing Ad5 particles was incubated at room temperature (about25° C.) or 35° C. with purification matrix having an amino acid sequenceof SEQ ID NO: 173, purification matrix control (having no specificityfor Ad5), or PBS.

At days 0, 3, 7, and 11, post incubation, the media was harvested andadministered to HEK293 cells. Flow cytometry was performed to evaluatethe ability of the Ad5 particles to infect HEK293 cells, as indicated bythe percentage of green fluorescing cells (labeled infectivity in FIG.15 ). The infectivity for each source of Ad5 particles was normalized tothat source’s day 0 infectivity. The infectivity of Ad5 particlesobtained from media incubated with purification matrix (labeledPurification Matrix) was higher than the infectivity of Ad5 particlesobtained from media incubated with purification matrix control (labeledMatrix Control), and media incubated with PBS (labeled PBS control)after 3, 7, and 11 days (FIG. 15 ). Media incubated in the presence ofpurification matrix was more infective than media incubated in thepresence of PBS (labeled control) at both room temperature and 35° C.(FIG. 16 ).

Example 14. Stabilization of Ad5 to Freeze-Thaw Cycles

Purified adenovirus type 5 (Ad5) particles carrying a green fluorescentprotein (GFP) transgene were formulated in PBS with or without theaddition of 10 µM purification matrix or Matrix control (no specificityfor Ad5). The samples were subjected to three freeze-thaw cycles (-80°C. to room temperature). Using flow cytometry, the ability of eachcomposition of Ad5 particles to infect HEK293 cells was quantified bycounting the percentage of green fluorescing cells and normalizing thepercentage of green fluorescing cells resulting from each composition toa control composition comprising Ad5 particles in the absence ofpurification matrix that were subjected to freeze-thaw cycles. Ad5particles incubated with Purification Matrix had 20-40% higherinfectivity compared to the Ad5-GFP particles incubated with a MatrixControl or PBS (labeled PBS control). (FIG. 17 ).

Example 15. Separation of Biologic From Contaminant Using TwoPurification Matrices

A purification matrix with a lower critical solution temperaturetransition temperature (T_(t)) is labeled with a blue fluorescent dyeand a purification matrix with an upper critical solution temperature(UCST) T_(t) is labeled with a red fluorescent dye. An environmentalfactor (e.g. NaCl) is added to the composition comprising bothpurification matrices to promote the formation of insoluble droplets ofboth purification matrices. Fluorescence imaging is performed toevaluate if each droplet contains a single purification matrix or bothpurification matrices.

The ability to form droplets containing a single purification matrixallows for separation of droplets of each purification matrix.

A first purification matrix that binds to a biologic and a secondpurification matrix that binds to a contaminant is added to acomposition containing a biologic and a contaminant. A first complexbetween the biologic and first purification matrix forms. A secondcomplex between the contaminant and second purification matrix forms. Anenvironmental factor is added to separate the first complex from thesecond complex.

Example 16. Purification of Recombinant IgG1 Using Purification Matricesand Tangential Flow Filtration (TFF)

Starting material (SM) containing ~4.8 g/L recombinant immunoglobulin 1(IgG1) secreted from CHO cells was contacted with about 100 µM ofpurification matrix. The capture domain of the purification matrix bindsto the Fc region of IgG1, resulting in a complex between purificationmatrix and IgG1. An environmental factor (0.6 M NaCl) was added toincrease the size of the complexes.

TFF was used to concentrate the complexes and to separate the complexesfrom impurities. The composition comprising the complexes was applied toa 13 cm² polyethersulfone (PES) membrane which contained 0.2 µm pores.The composition was concentrated 2X and washed with a phosphate-bufferedsaline (PBS) wash buffer containing 0.6 M NaCl to remove impurities fromthe complexes. An elution buffer (7 diavolumes) was applied to thefilter (50 mM sodium citrate, pH 3, 0.6 M NaCl) to separate the IgG fromthe purification matrix. trigger release of the captured, concentratedand purified IgG1. The purity of the IgG was assessed by an SDS-PAGEgel, stained with a Coomassie dye (FIG. 18 ). The SDS-PAGE gel shows thehigh purity and recovery of the eluted IgG1.

High flux and transmembrane pressure (TMP) were maintained throughoutthe run (FIG. 19 ), indicating that the use of TFF enables a reductionin the time required to concentrate and separate a biologic from animpurity.

Size exclusion chromatography was performed to evaluate the purity ofthe eluted IgG1 product. The IgG1 product was applied to a TSKgel3000sWXL column, and a mobile phase (i.e. 50 mM phosphate, 100 mM NaCl,at pH 7) was applied to the column at a 0.4 mL/min flow rate. The pureIgG1 was detected by measuring the absorbance at 230 nm. In comparisonto the unpurified IgG1 composition, the pure IgG1 was > 98 % pure.Furthermore, the IgG1 product contained < 1.5 % of aggregated IgG1 (FIG.20 ).

The purification process described above was repeated with antibodies ofdifferent subclasses (IgG2, IgG4, IgG1). The purity of antibodiespurified from the aforementioned method was compared to antibodiespurified by protein A chromatography (FIG. 21 ). Antibodies purifiedwith a purification matrix exhibited less contaminant host cell proteins(HCP) than antibodies purified by protein A chromatography. Host cellproteins were quantified with a Cygnus™ CHO host cell protein (HCP)ELISA detection kit.

The purification matrices also provided higher productivity than theprotein A resins used in protein A affinity chromatography (FIG. 22 ).Productivity was calculated as grams of antibody per unit of material(resin or purification matrix) per hour.

Example 17. Purification of IgG1, IgG2, and IgG4 Using a PurificationMatrix

The effect of purification matrix on yield and purity of IgG1, IgG2, andIgG4 was evaluated. The following purification methods (a) and (b) wereevaluated:

Purification method (a): IgG1, IgG2, or IgG4 from the supernatants ofCHO cells containing secreted IgG1, IgG2, or IgG4 was purified byincubating the supernatant with the purification matrix of Example 16 toform complexes, increasing the size of the complexes by the addition ofa first environmental factor (i.e., 0.6 M NaCl), separating thecomplexes from impurities using centrifugation, and eluting the IgG1,IgG2, or IgG4 from the purification matrix with a second environmentalfactor (i.e., 50 mM sodium citrate, pH 4).

Purification method (b): IgG1, IgG2, or IgG4 from the supernatants ofCHO cells containing secreted IgG1, IgG2, or IgG4 was purified byincubating the supernatant with MAbSelect™ SuRe affinity chromatographyresin (an alkali-stabilized protein A-derived ligand), washing with 140mM NaCl, 10 mM phosphate buffer, and 3 mM KCl, pH 7.4, and eluting with50 mM citrate buffer at pH 3.

Ultraviolet-visible spectrophotometry (UV-Vis) and size exclusionchromatography (SEC) were utilized to determine the yield of IgG1, IgG2,and IgG4 produced according to purification method (a) or purificationmethod (b). Size exclusion chromatography was used to determine theamount of high molecular weight (HMW) and low molecular weight (LMW)impurities in each sample. Purification method (a) resulted in purifiedimmunoglobulin that is more concentrated than that produced bypurification method (b). Table 3 shows the yield and percentage of HMWand LMW impurities present in the purified immunoglobulin compositionsof purification method (a) and purification (b). The size exclusionchromatography area under the curve (SEC AUC) concentration and yieldwere obtained by normalizing the area under the curve (AUC) from SECobtained from method (a) to that of method (2). Molecule-optimizeddownstream process values are shown in parentheses.

TABLE 3 Yield and concentration of Immunoglobulins by method (a) versusmethod (b) Cell culture harvest (CCH )titer (g/L) Concentration (g/L) asdetermined by UV-Vis Plate Yield as determined by UV-Vis (%) SEC AUCConcentration (Normalized) SEC AUC Yield (Normalized) SEC ChromatogramAUC Analysis Peak Containing HMW (%) Peak containing antibody (%) PeakContaining LMW (%) IgG 2 (2.8) (99.6) (0.0) Method (b) 3.17 1.20 22.71.00 1.00 1.79 97.99 0.22 Method (a) 4.71 74.0 3.65 3.23 3.17 96.57 0.14IgG1 (2.7) (97.3) (0.0) Method (b) 0.87 0.39 26.9 1.00 1.00 1.55 98.450.00 Method (a) 1.32 75.6 3.50 2.30 2.54 97.25 0.21 IgG4 (0.8) (99.2)(0.0) Method (b) 0.70 0.39 33.4 1.00 1.00 0.47 99.53 0.00 Method (a)1.39 98.9 2.33 3.63 1.07 98.82 0.11

Example 18 Purification of Multiple AAV Serotypes Using a PurificationMatrix

Recombinant AAV particles, including AAV1, AAV2, AAV6, AAV8, and AAV9particles, packaging a tdTomato transgene, were produced in a producercell line (e.g., HEK293) according to standard protocols. The cells werelysed, and centrifuged to remove cellular debris. The cellularsupernatant was contacted with a purification matrix having an aminoacid sequence of SEQ ID NO: 172 for a period of time to allow complexformation. An environmental factor (e.g. 0.5 M - 2 M NaCl, MgCl₂, orCaCl₂) was then applied to increase the size of the complexes. Thepurification matrix, environmental factor, and cellular supernatant wereincubated at room temperature for 15 minutes. Subsequently, thepurification matrix, environmental factor, and cellular supernatant wereconcentrated (5-50 fold) using a 13 cm² hollow filter (0.2 µm poresize). Six wash diavolumes were performed using phosphate buffersolution and sodium chloride. This protocol allowed for separation ofthe purification matrix, with AAVs bound from impurities on the basis ofsize. The AAVs were eluted from the purification matrix by applying abuffer (e.g., the second environmental factor). The purification matrixwas collected from the retentate. Various buffers were evaluated asshown in Table 5. The AAVs are then titered and frozen at -80° C. forfuture use.

TABLE 5 Buffers employed as the “second environmental factor”Environmental Factor Composition of Second Environmental Factor A 0.5 MArginine (pH = 2); 0.6 M NaCl B 0.1 M Glycine (pH = 2); 0.6 M NaCl C 0.1M Glycine (pH = 2); 0.6 M MgCl₂ D 0.1 M Glycine (pH = 2); 0.6 M CaCl₂

Quantitative real-time polymerase chain reaction (qPCR) was utilized toevaluate the amount of AAV captured from solution and the amount of AAVobtained after elution. The purification matrix captured greater than99% of AAV particles of multiple serotypes (AAV1, AAV2, AAV6, AAV8, andAAV9) (FIG. 23 ). Each buffer evaluated eluted over 65 % of bound AAVparticles in a single diavolume (FIG. 24 ).

The purification matrix was recycled after elution of the AAV particlesto determine whether or not it could be utilized for futurepurifications. Recycling was performed by incubating the purificationmatrix at 95° C. for 5 minutes or soaking the purification matrix in 1 MNaOH or 6 M guanidine hydrochloride for five minutes.

As Table 6 shows, the purification matrix can be regenerated andutilized for repeated capture. After five cycles ofpurification/regeneration, the purification matrix captures 98 % of AAV.

TABLE 6 Recycling of Purification Matrix # of times purification matrixhas been used Captured viral genome (vg) / milliliter (mL) % Capture 18.46 × 10¹⁰ 97 2 8.60 × 10¹⁰ 98 3 8.61 × 10¹⁰ 99 4 8.57 × 10¹⁰ 98 5 8.53× 10¹⁰ 98

Sodium dodecycl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was utilized to evaluate the purity of the eluted AAV samples. The gelswere then silver stained, to visualize any contaminants in the sample.As shown in FIG. 25 , the major AAV structural proteins Vp1, Vp2, andVp3 were visible on the gel, but no other major bands were observed. AWestern Blot confirmed the presence of capsid proteins in the samplethat had been eluted from the purification matrix (FIG. 26 ).Furthermore, after the AAV particles were captured by the purificationmatrix, no AAV particles remained in the capture supernatant (CaptureSup of FIG. 26 ). Taken together, this data indicates that, afterelution from the purification matrix, substantially all contaminantshave been removed from the sample, and the isolated AAV has a highdegree of purity.

In a subsequent experiment, it was evaluated whether the purificationmatrix is able to capture full capsids, empty capsids, or both. Totalnumber of AAV capsids present in samples eluted from the purificationmatrix was estimated using an ELISA-based assay. The eluted samples werealso evaluated using qPCR, to determine the number of viral genomes. AqPCR:ELISA value was used to approximate the ratio of full capsidsrelative to total capsids. As shown in Table 7, the purification matrixenriched for full capsids.

TABLE 7 Enrichment for Full AAV Capsid Method Starting Quantity Capture% Elution Quantity Change in Full % qPCR 5.78 × 10¹⁰ 99.2 5.73 × 10¹⁰33% → 70% ELISA 1.76 × 10¹¹ 99.9 8.14 × 10¹⁰

The eluted AAV samples were also assayed to determine whether theymaintained infectivity after purification. AAV8 carrying a tdTomatotransgene was administered to HEK293 cells (10,000 cells/ well) inculture at a multiplicity of infection (MOI) 1 × 10⁶ or 1 × 10⁷. After48 of incubation, the cells were visualized for tdTomato fluorescenceusing fluorescence microscopy. As shown in FIG. 27A, AAV purified by thepurification matrix (ViraTag™) infected cells to a similar extent asAAVs purified according to standard protocols (Pos Ctrl). This data wasquantified, as shown in FIG. 27B. There was no statistically significantdifference between infectivity levels of the AAV8 purified by standardprotocols (Pos Ctrl) and the AAV8 purified by the purification matrix(ViraTag™). Accordingly, this data shows that AAV particles purifiedusing the tested purification matrix (ViraTag™) retain high levels ofinfectivity.

NUMBERED EMBODIMENTS

Notwithstanding the appended claims, the disclosure sets forth thefollowing numbered embodiments:

Method of Purifying a Biologic From a Contaminant

1. A method of purifying a biologic comprising contacting the biologicwith a protein-based purification matrix;

-   wherein the biologic binds to the purification matrix to form a    complex;-   wherein the size of the complex is increased by a first    environmental factor;-   wherein the complex is separated from at least one contaminant on    the basis of size; and-   wherein the biologic is separated from the purification matrix by a    second environmental factor.

2. The method of embodiment 1, wherein the purification matrix comprises(i) a capture domain which binds to the biologic, and (ii) a polypeptidewith phase behavior, wherein the capture domain is coupled to thepolypeptide with phase behavior.

3. The method of embodiment 2, wherein the capture domain is coupled tothe polypeptide with phase behavior via a linker.

4. The method of embodiment 3, wherein the linker is a peptide linker.

5. The method of embodiment 4, wherein the peptide linker comprises aprotease cleavage site.

6. The method of embodiment 3, wherein the linker is a chemical linker.

7. The method of embodiment 1, wherein the purification matrix comprisesa fusion protein comprising (i) a capture domain which binds to thebiologic and (ii) a polypeptide with phase behavior.

8. The method of any one of embodiments 2-7, wherein the polypeptidewith phase behavior is a resilin-like polypeptide.

9. The method of any one of embodiments 2-7, wherein the polypeptidewith phase behavior is an elastin-like polypeptide.

10. The method of any one of 2-7 or 9, wherein the polypeptide withphase behavior is a polymer containing a pentapeptide repeat having thesequence (Val-Pro-Gly-Xaa-Gly)_(n) (SEQ ID NO: 10), or a randomized,scrambled analog thereof; wherein Xaa can be any amino acid exceptproline.

11. The method of embodiment 10, wherein n is an integer from 1 to 360,inclusive of endpoints.

12. The method of any one of embodiments 2-7 or 9, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GRGDSPY)n (SEQ ID NO: 1)-   b. (GRGDSPH)n (SEQ ID NO: 2)-   c. (GRGDSPV)_(n) (SEQ ID NO: 3)-   d. (GRGDSPYG)_(n) (SEQ ID NO: 4)-   e. (RPLGYDS)_(n) (SEQ ID NO: 5)-   f. (RPAGYDS)_(n) (SEQ ID NO: 6)-   g. (GRGDSYP)_(n) (SEQ ID NO: 7)-   h. (GRGDSPYQ)_(n) (SEQ ID NO: 8)-   i. (GRGNSPYG)_(n) (SEQ ID NO: 9)-   j. (GVGVP)_(n) (SEQ ID NO: 11);-   k. (GVGVPGLGVPGVGVPGLGVPGVGVP)_(m) (SEQ ID NO: 12);-   1. (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 13);-   m. (GVGVPGWGVPGVGVPGWGVPGVGVP)_(m) (SEQ ID NO: 14);-   n. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m (SEQ ID NO: 15);-   o. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m (SEQ ID NO: 16);    and-   p. (GAGVPGVGVPGAGVPGVGVPGAGVP)_(m) (SEQ ID NO: 17);-   or a randomized, scrambled analog thereof;-   wherein:    -   n is an integer in the range of 20-360, inclusive of endpoints;        and    -   m is an integer in the range of 4-25, inclusive of endpoints.

13. The method of any one of embodiments 2-7 or 9, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVP)_(m) (SEQ ID NO: 52);-   (b) (ZZPXXXXGZ)_(m) (SEQ ID NO: 57);-   (c) (ZZPXGZ)_(m) (SEQ ID NO: 58);-   (d) (ZZPXXGZ)_(m) (SEQ ID NO: 59); or-   (e) (ZZPXXXGZ)_(m) (SEQ ID NO: 60),-   wherein m is an integer between 10 and 160, inclusive of endpoints,    wherein X if present is any amino acid except proline or glycine,    and wherein Z if present is any amino acid.

14. The method of any one of embodiments 2-7 or 9, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 53); or-   (b) (GVGVPGVGVPGLGVPGVGVPGVGVP)_(m) (SEQ ID NO: 55);-   wherein m is an integer between 2 and 32, inclusive of endpoints.

15. The method of any one of embodiments 2-7 or 9, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 193), wherein m is 8    or 16;-   (b) (GVGVPGAGVP)_(m) (SEQ ID NO: 54), wherein m is an integer    between 5 and 80, inclusive of endpoints; or-   (c) (GXGVP)_(m) (SEQ ID NO: 56), wherein m is an integer between 10    and 160, inclusive of endpoints, and wherein X for each repeat is    independently selected from the group consisting of glycine,    alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,    tryptophan, lysine, arginine, aspartic acid, glutamic acid, and    serine.

16. The method of any one of embodiments 2-15, wherein the capturedomain comprises the sequence of any one of SEQ ID NO: 24-49, 62-148,167-171 or a sequence having at least 1, at least 2, at least 3, atleast 4, or at least 5 mutations relative thereto.

17. The method of any one of embodiments 2-15, wherein the binding ofthe biologic to the purification matrix is reversible.

18. The method of any one of embodiments 2-15, wherein the binding ofthe biologic to the purification matrix is non-covalent.

19. The method of any one of embodiments 2-15, wherein the binding ofthe biologic to the purification matrix is covalent.

20. The method of any one of embodiments 1-19, wherein the biologic is alipid, a lipopolysaccharide, a cell, a protein, a nucleic acid, acarbohydrate, or a viral particle.

21. The method of embodiment 20, wherein the biologic is a cell.

22. The method of embodiment 21, wherein the cell is a bacterial cell, ayeast cell, or a mammalian cell.

23. The method of embodiment 21 or 22, wherein the cell is a stem cell,a bone cell, a blood cell, a muscle cell, a fat cell, a skin cell, anerve cell, an endothelial cell, a sex cell, a pancreatic cell, or acancer cell.

24. The method of embodiment 21 or 22, wherein the cell is an immunecell.

25. The method of embodiment 24, wherein the immune cell is a T cell, aB cell, a NK cell, a peripheral blood mononuclear cell, or a neutrophil.

26. The method of embodiment 25, wherein the cell is a T cell expressinga chimeric antigen receptor (CAR).

27. The method of embodiment 20, wherein the nucleic acid is a DNA or anRNA.

28. The method of embodiment 17, wherein the viral particle is anadenovirus particle, an adeno-associated virus (AAV) particle, alentivirus particle, a retrovirus particle, a poxvirus particle, ameasles virus particle, or a herpesvirus particle.

29. The method of any one of embodiments 1-28, wherein the biologic hasa diameter between 1 nm and 100 µm, inclusive of the endpoints.

30. The method of embodiment 29, wherein the biologic has a diameterbetween 1 nm and 100 nm, inclusive of the endpoints.

31. The method of embodiment 29, wherein the biologic has a diameterbetween 100 nm and 1 µm, inclusive of the endpoints.

32. The method of embodiment 29, wherein the biologic has a diameterbetween 1 µm and 50 µm, inclusive of the endpoints.

33. The method of embodiment 29, wherein the biologic has a diameterbetween 50 µm and 100 µm, inclusive of the endpoints.

34. The method of any one of embodiments 1-33, wherein the method iscompleted in about 0.5 to about 24 hours.

35. The method of embodiment 34, wherein the method is completed inabout 0.5 to about 8 hours.

36. The method of embodiment 34, wherein the method is completed inabout 2 to about 6 hours.

37. The method of any one of embodiments 1-36, wherein the separation ofthe complex from the at least one contaminant can be observed visuallywith an unaided eye.

38. The method of any one of embodiments 1-37, wherein the increase inthe size of the complex is at least a 2-fold increase.

39. The method of embodiment 38, wherein the increase in the size of thecomplex is at least a 10-fold increase.

40. The method of embodiment 38, wherein the increase in the size of thecomplex is at least a 25-fold increase.

41. The method of any one of embodiments 38-40, wherein the increase insize is an increase in the mass of the complex.

42. The method of any one of embodiments 38-40, wherein the increase insize is an increase in the diameter of the complex.

43. The method of any one of embodiments 1-42, wherein the firstenvironmental factor comprises one or more of:

-   a. a change in one or more of temperature, pH, salt concentration,    concentration of the purification matrix, concentration of the    biologic, or pressure;-   b. the addition of one or more surfactants, cofactor, vitamin,    molecular crowding agents, reducing agents, oxidizing agents,    enzymes, or denaturing agents; or-   c. the application of electromagnetic or acoustic waves.

44. The method of any one of embodiments 1-42, wherein the secondenvironmental factor comprises one or more of:

-   a. a change in one or more of temperature, pH, salt concentration,    concentration of the purification matrix, concentration of the    biologic, or pressure;-   b. the addition of one or more surfactants, molecular crowding    agents, reducing agents, enzymes, denaturing agents, cofactor,    vitamin, or oxidizing agents; or-   c. the application of electromagnetic or acoustic waves.

45. The method of any one of embodiments 1-44, wherein the separation onthe basis of size is performed using tangential flow filtration,membrane chromatography, analytical ultracentrifugation, highperformance liquid chromatography, membrane chromatography, normal flowfiltration, acoustic wave separation, centrifugation, counterflowcentrifugation, and fast protein liquid chromatography.

46. The method of any one of embodiments 1-45, wherein the at least onecontaminant is selected from a solvent, an endotoxin, a protein, apeptide, a nucleic acid, and a carbohydrate.

47. The method of any one of embodiments 1-46, wherein the purificationyield of the biologic is at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

48. The method of any one of embodiments 1-47, wherein the biologic ispurified to at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% purity.

Method of Removing a Contaminant From a Composition Comprising aBiologic

1a. A method of removing a contaminant from a composition comprising abiologic, the method comprising contacting the contaminant with aprotein-based purification matrix;

-   wherein the contaminant binds to the matrix to form a complex;-   wherein the size of the complex is increased by a first    environmental factor;-   wherein the complex is separated from the biologic on the basis of    size; and-   wherein the contaminant is separated from the matrix by a second    environmental factor.

2a. The method of embodiment 1a, wherein the purification matrixcomprises (i) a capture domain which binds to the contaminant, and (ii)a polypeptide with phase behavior, wherein the capture domain is coupledto the polypeptide with phase behavior.

3a. The method of embodiment 2a, wherein the capture domain is coupledto the polypeptide with phase behavior via a linker.

4a. The method of embodiment 3a, wherein the linker is a peptide linker.

5a. The method of embodiment 4a, wherein the peptide linker comprises aprotease cleavage site.

6a. The method of embodiment 3a, wherein the linker is a chemicallinker.

7a. The method of embodiment 1a, wherein the purification matrixcomprises a fusion protein comprising (i) a capture domain which bindsto the contaminant and (ii) a polypeptide with phase behavior.

8a. The method of any one of embodiments 2a-7a, wherein the polypeptidewith phase behavior is a resilin-like polypeptide.

9a. The method of any one of embodiments 2a-7a, wherein the polypeptidewith phase behavior is an elastin-like polypeptide.

10a. The method of any one of 2a-7a or 9a, wherein the polypeptide withphase behavior is a polymer containing a pentapeptide repeat having thesequence (Val-Pro-Gly-Xaa-Gly)_(n) (SEQ ID NO: 10), or a randomized,scrambled analog thereof; wherein Xaa can be any amino acid exceptproline.

11a. The method of embodiment 10a, wherein n is an integer from 1 to360, inclusive of endpoints.

12a. The method of any one of embodiments 2a-7a or 9a, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GRGDSPY)n (SEQ ID NO: 1)-   b. (GRGDSPH)n (SEQ ID NO: 2)-   c. (GRGDSPV)_(n) (SEQ ID NO: 3)-   d. (GRGDSPYG)_(n) (SEQ ID NO: 4)-   e. (RPLGYDS)_(n) (SEQ ID NO: 5)-   f. (RPAGYDS)_(n) (SEQ ID NO: 6)-   g. (GRGDSYP)_(n) (SEQ ID NO: 7)-   h. (GRGDSPYQ)_(n) (SEQ ID NO: 8)-   i. (GRGNSPYG)_(n) (SEQ ID NO: 9)-   j. (GVGVP)_(n) (SEQ ID NO: 11);-   k. (GVGVPGLGVPGVGVPGLGVPGVGVP)_(m) (SEQ ID NO: 12);-   1. (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 13);-   m. (GVGVPGWGVPGVGVPGWGVPGVGVP)_(m) (SEQ ID NO: 14);-   n. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m (SEQ ID NO: 15);-   o. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m (SEQ ID NO: 16);    and-   p. (GAGVPGVGVPGAGVPGVGVPGAGVP)_(m) (SEQ ID NO: 17);-   or a randomized, scrambled analog thereof;-   wherein:    -   n is an integer in the range of 20-360, inclusive of endpoints;        and    -   m is an integer in the range of 4-25, inclusive of endpoints.

13a. The method of any one of embodiments 2a-7a or 9a, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GVGVP)_(m) (SEQ ID NO: 52);-   b. (ZZPXXXXGZ)_(m) (SEQ ID NO: 57);-   c. (ZZPXGZ)_(m) (SEQ ID NO: 58);-   d. (ZZPXXGZ)_(m) (SEQ ID NO: 59); or-   e. (ZZPXXXGZ)_(m) (SEQ ID NO: 60),-   wherein m is an integer between 10 and 160, inclusive of endpoints,    wherein X if present is any amino acid except proline or glycine,    and wherein Z if present is any amino acid.

14a. The method of any one of embodiments 2a-7a or 9a, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 53); or-   (b) (GVGVPGVGVPGLGVPGVGVPGVGVP)_(m) (SEQ ID NO: 55);-   wherein m is an integer between 2 and 32, inclusive of endpoints.

15a. The method of any one of embodiments 2a-7a or 9a, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 193), wherein m is 8    or 16;-   (b) (GVGVPGAGVP)_(m) (SEQ ID NO: 54), wherein m is an integer    between 5 and 80, inclusive of endpoints; or-   (c) (GXGVP)_(m) (SEQ ID NO: 56), wherein m is an integer between 10    and 160, inclusive of endpoints, and wherein X for each repeat is    independently selected from the group consisting of glycine,    alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,    tryptophan, lysine, arginine, aspartic acid, glutamic acid, and    serine.

16a. The method of any one of embodiments 2a-15a, wherein the capturedomain comprises the sequence of any one of SEQ ID NO: 24-49, 62-148, or167-171 or a sequence having at least 1, at least 2, at least 3, atleast 4, or at least 5 mutations relative thereto.

17a. The method of any one of embodiments 1a-16a, wherein the binding ofthe contaminant to the purification matrix is reversible.

18a. The method of any one of embodiments 1a-16a, wherein the binding ofthe contaminant to the purification matrix is non-covalent.

19a. The method of any one of embodiments 1a-16a, wherein the binding ofthe contaminant to the purification matrix is covalent.

20a. The method of any one of embodiments 1a-19a, wherein the biologicis a lipid, lipopolysaccharide, cell, a protein, a nucleic acid, acarbohydrate, or a viral particle.

21a. The method of embodiment 20a, wherein the biologic is a cell.

22a. The method of embodiment 32a, wherein the cell is a bacterial cell,a yeast cell, or a mammalian cell.

23a. The method of embodiment 21a or 22a, wherein the cell is a stemcell, a bone cell, a blood cell, a muscle cell, a fat cell, a skin cell,a nerve cell, an endothelial cell, a sex cell, a pancreatic cell, or acancer cell.

24a. The method of embodiments 21a or 22a, wherein the cell is an immunecell.

25a. The method of embodiment 24a, wherein the immune cell is a T cell,a B cell, a NK cell, a peripheral blood mononuclear cell, or aneutrophil.

26a. The method of embodiment 25a, wherein the cell is a T cellexpressing a chimeric antigen receptor (CAR).

27a. The method of embodiment 20a, wherein the nucleic acid is a DNA oran RNA.

28a. The method of embodiment 20a, wherein the viral particle is anadenovirus particle, an adeno-associated virus (AAV) particle, alentivirus particle, a retrovirus particle, a poxvirus particle, ameasles virus particle, or a herpesvirus particle.

29a. The method of any one of embodiments 1a-28a, wherein the biologichas a diameter between 1 nm and 100 µm, inclusive of the endpoints.

30a. The method of embodiment 29a, wherein the biologic has a diameterbetween 1 nm and 100 nm, inclusive of the endpoints.

31a. The method of embodiment 29a, wherein the biologic has a diameterbetween 100 nm and 1 µm, inclusive of the endpoints.

32a. The method of embodiment 29a, wherein the biologic has a diameterbetween 1 µm and 50 µm, inclusive of the endpoints.

33a. The method of embodiment 29a, wherein the biologic has a diameterbetween 50 µm and 100 µm, inclusive of the endpoints.

34a. The method of any one of embodiments 1a-33a, wherein the method iscompleted in about 0.5 to about 24 hours.

35a. The method of embodiment 34a, wherein the method is completed inabout 0.5 to about 8 hours.

36a. The method of embodiment 34a, wherein the method is completed inabout 2 to about 6 hours.

37a. The method of any one of embodiments 1a-36a, wherein the separationof the complex from the composition comprising a biologic can beobserved visually with an unaided eye.

38a. The method of any one of embodiments 1a-37a, wherein the increasein the size of the complex is at least a 2-fold increase.

39a. The method of embodiment 80a, wherein the increase in the size ofthe complex is at least a 10-fold increase.

40a. The method of embodiment 39a, wherein the increase in the size ofthe complex is at least a 25-fold increase.

41a. The method of any one of embodiments 38a-40a, wherein the increasein size is an increase in the mass of the complex.

42a. The method of any one of embodiments 38a-40a, wherein the increasein size is an increase in the diameter of the complex.

43a. The method of any one of embodiments 1a-42a, wherein the firstenvironmental factor comprises one or more of:

-   a. a change in one or more of temperature, pH, salt concentration,    concentration of the purification matrix, concentration of the    biologic, or pressure;-   b. the addition of one or more surfactants, molecular crowding    agents, reducing agents, oxidizing agents, enzymes, cofactor,    vitamin, or denaturing agents; or-   c. the application of electromagnetic or acoustic waves.

44a. The method of any one of embodiments 1a-42a, wherein the secondenvironmental factor comprises one or more of:

-   a. a change in one or more of temperature, pH, salt concentration,    concentration of the purification matrix, concentration of the    biologic, or pressure;-   b. the addition of one or more surfactants, molecular crowding    agents, reducing agents, oxidizing agents, enzymes, cofactor,    vitamin, or denaturing agents; or c. the application of    electromagnetic or acoustic waves.

45a. The method of any one of embodiments 1a-44a, wherein the separationon the basis of size is performed using tangential flow filtration,analytical ultracentrifugation, membrane chromatography, highperformance liquid chromatography, normal flow filtration, acoustic waveseparation, centrifugation, counterflow centrifugation, and fast proteinliquid chromatography. 46a. The method of any one of embodiments 1a-45a,wherein the contaminant is selected from a solvent, an endotoxin, aprotein, a peptide, a nucleic acid, and a carbohydrate.

47a. The method of any one of embodiments 1a-46a, wherein at least 70%,at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% of the contaminant is removed.

Method of Purifying a Biologic From a Contaminant

1b. A method of purifying a biologic, the method comprising contactingthe biologic with a protein-based purification matrix;

-   wherein the biologic binds to the matrix to form a complex;-   wherein the size of the complex is increased;-   wherein the complex is separated from at least one contaminant on    the basis of size; and-   wherein the biologic is separated from the matrix by an    environmental factor.

2b. The method of embodiment 1b, wherein the purification matrixcomprises (i) a capture domain which binds to the biologic, and (ii) apolypeptide with phase behavior, wherein the capture domain is coupledto the polypeptide with phase behavior.

3b. The method of embodiment 2b, wherein the capture domain is coupledto the polypeptide with phase behavior via a linker.

4b. The method of embodiment 3b, wherein the linker is a peptide linker.

5b. The method of embodiment 4b, wherein the peptide linker comprises aprotease cleavage site.

6b. The method of embodiment 3b, wherein the linker is a chemicallinker.

7b. The method of embodiment 1b, wherein the purification matrixcomprises a fusion protein comprising (i) a capture domain which bindsto the biologic and (ii) a polypeptide with phase behavior.

8b. The method of any one of embodiments 2b-7b, wherein the polypeptidewith phase behavior is a resilin-like polypeptide.

9b. The method of any one of embodiments 2b-7b, wherein the polypeptidewith phase behavior is an elastin-like polypeptide.

10b. The method of any one of 2b-7b or 9b, wherein the polypeptide withphase behavior is a polymer containing a pentapeptide repeat having thesequence (Val-Pro-Gly-Xaa-Gly)_(n) (SEQ ID NO: 10), or a randomized,scrambled analog thereof; wherein Xaa can be any amino acid exceptproline.

11b. The method of embodiment 10b, wherein n is an integer from 1 to360, inclusive of endpoints.

12b. The method of any one of embodiments 2b-7b or 9b, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GRGDSPY)_(n) (SEQ ID NO: 1)-   b. (GRGDSPH)_(n) (SEQ ID NO: 2)-   c. (GRGDSPV)_(n) (SEQ ID NO: 3)-   d. (GRGDSPYG)_(n) (SEQ ID NO: 4)-   e. (RPLGYDS)_(n) (SEQ ID NO: 5)-   f. (RPAGYDS)_(n) (SEQ ID NO: 6)-   g. (GRGDSYP)_(n) (SEQ ID NO: 7)-   h. (GRGDSPYQ)_(n) (SEQ ID NO: 8)-   i. (GRGNSPYG)_(n) (SEQ ID NO: 9)-   j. (GVGVP)_(n) (SEQ ID NO: 11);-   k. (GVGVPGLGVPGVGVPGLGVPGVGVP)_(m) (SEQ ID NO: 12);-   l. (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 13);-   m. (GVGVPGWGVPGVGVPGWGVPGVGVP)_(m) (SEQ ID NO: 14);-   n. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)_(m) (SEQ ID NO:    15);-   o. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)_(m) (SEQ ID NO:    16); and-   p. (GAGVPGVGVPGAGVPGVGVPGAGVP)_(m) (SEQ ID NO: 17);-   or a randomized, scrambled analog thereof; wherein:    -   n is an integer in the range of 20-360, inclusive of endpoints;        and    -   m is an integer in the range of 4-25, inclusive of endpoints.

13b. The method of any one of embodiments 2b-7b or 9b, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GVGVP)_(m) (SEQ ID NO: 52);-   b. (ZZPXXXXGZ)_(m) (SEQ ID NO: 57);-   c. (ZZPXGZ)_(m) (SEQ ID NO: 58);-   d. (ZZPXXGZ)_(m) (SEQ ID NO: 59); or-   e. (ZZPXXXGZ)_(m) (SEQ ID NO: 60),-   wherein m is an integer between 10 and 160, inclusive of endpoints,    wherein X if present is any amino acid except proline or glycine,    and wherein Z if present is any amino acid.

14b. The method of any one of embodiments 2b-7b or 9b, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 53); or-   (b) (GVGVPGVGVPGLGVPGVGVPGVGVP)_(m) (SEQ ID NO: 55);-   wherein m is an integer between 2 and 32, inclusive of endpoints.

15b. The method of any one of embodiments 2b-7b or 9b, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 193), wherein m is 8    or 16;-   (b) (GVGVPGAGVP)_(m) (SEQ ID NO: 54), wherein m is an integer    between 5 and 80, inclusive of endpoints; or-   (c) (GXGVP)_(m) (SEQ ID NO: 56), wherein m is an integer between 10    and 160, inclusive of endpoints, and wherein X for each repeat is    independently selected from the group consisting of glycine,    alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,    tryptophan, lysine, arginine, aspartic acid, glutamic acid, and    serine.

16b. The method of any one of embodiments 2b-15b, wherein the capturedomain comprises the sequence of any one of SEQ ID NO: 24-49, 62-148, or167-171 or a sequence having at least 1, at least 2, at least 3, atleast 4, or at least 5 mutations relative thereto.

17b. The method of any one of embodiments 1b-16b, wherein the binding ofthe biologic to the purification matrix is reversible.

18b. The method of any one of embodiments 1b-16b, wherein the binding ofthe biologic to the purification matrix is non-covalent.

19b. The method of any one of embodiments 1b-16b, wherein the binding ofthe biologic to the purification matrix is covalent.

20b. The method of any one of embodiments 1b-19b, wherein the biologicis a lipid, lipopolysaccharide, cell, a protein, a nucleic acid, acarbohydrate, or a viral particle.

21b. The method of embodiment 20b, wherein the biologic is a cell.

22b. The method of embodiment 21b, wherein the cell is a bacterial cell,a yeast cell, or a mammalian cell.

23b. The method of embodiment 21b or 22b, wherein the cell is a stemcell, a bone cell, a blood cell, a muscle cell, a fat cell, a skin cell,a nerve cell, an endothelial cell, a sex cell, a pancreatic cell, or acancer cell.

24b. The method of any one of embodiments 21b or 22b, wherein the cellis an immune cell. 25b. The method of embodiment 24b, wherein the immunecell is a T cell, a B cell, a NK cell, a peripheral blood mononuclearcell, or a neutrophil.

26b. The method of embodiment 25b, wherein the cell is a T cellexpressing a chimeric antigen receptor (CAR).

27b. The method of embodiment 20b, wherein the nucleic acid is a DNA oran RNA.

28b. The method of embodiment 20b, wherein the virus is an adenovirusparticle, an adeno-associated virus (AAV) particle, a lentivirusparticle, a retrovirus particle, a poxvirus particle, a measles virusparticle, or a herpesvirus particle.

29b. The method of any one of embodiments 1b-28b, wherein the biologichas a diameter between 1 nm and 100 µm, inclusive of the endpoints.

30b. The method of embodiment 29b, wherein the biologic has a diameterbetween 1 nm and 100 nm, inclusive of the endpoints.

31b. The method of embodiment 29b, wherein the biologic has a diameterbetween 100 nm and 1 µm, inclusive of the endpoints.

32b. The method of embodiment 29b, wherein the biologic has a diameterbetween 1 µm and 50 µm, inclusive of the endpoints.

33b. The method of embodiment 29b, wherein the biologic has a diameterbetween 50 µm and 100 µm, inclusive of the endpoints.

34b. The method of any one of embodiments 1b-33b, wherein the method iscompleted in about 0.5 to about 24 hours.

35b. The method of embodiment 34b, wherein the method is completed inabout 0.5 to about 8 hours.

36b. The method of embodiment 34b, wherein the method is completed inabout 2 to about 6 hours.

37b. The method of any one of embodiments 1b-36b, wherein the separationof the complex from the at least one contaminant can be observedvisually with an unaided eye.

38b. The method of any one of embodiments 1b-37b, wherein the increasein the size of the complex is at least a 2-fold increase.

39b. The method of embodiment 38b, wherein the increase in the size ofthe complex is at least a 10-fold increase.

40b. The method of embodiment 39b, wherein the increase in the size ofthe complex is at least a 25-fold increase.

41b. The method of any one of embodiments 38b-40b, wherein the increasein size is an increase in the mass of the complex.

42b. The method of any one of embodiments 38b-40b, wherein the increasein size is an increase in the diameter of the complex.

43b. The method of any one of embodiments 1b-42b, wherein theenvironmental factor comprises one or more of:

-   a. a change in one or more of temperature, pH, salt concentration,    concentration of the purification matrix, concentration of the    biologic, or pressure;-   b. the addition of one or more surfactants, molecular crowding    agents, reducing agents, oxidizing agents, enzymes, cofactor,    vitamin, or denaturing agents; or-   c. the application of electromagnetic or acoustic waves.

44b. The method of any one of embodiments 1b-43b, wherein the separationon the basis of size is performed using tangential flow filtration,analytical ultracentrifugation, membrane chromatography, highperformance liquid chromatography, normal flow filtration, acoustic waveseparation, centrifugation, counterflow centrifugation, and fast proteinliquid chromatography.

45b. The method of any one of embodiments 1b-44b, wherein the at leastone contaminant is selected from a solvent, an endotoxin, a protein, apeptide, a nucleic acid, and a carbohydrate. 46b. The method of any oneof embodiments 1b-45b, wherein the purification yield of the biologic isat least 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%.

47b. The method of any one of embodiments 1b-46b, wherein the biologicis purified to at least 70%, at least 80%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% purity.

Method of Separating a First Biologic From a Second Biologic

1c. A method of separating a first biologic from a second biologic, themethod comprising

-   contacting the first biologic with a first protein-based    purification matrix and contacting the second biologic with a second    protein-based purification matrix; wherein the first biologic binds    to the first purification matrix to form a first complex; wherein    the second biologic binds to the second purification matrix to form    a second complex; and-   separating the first biologic from the second biologic by applying    an environmental factor.

2c. The method of embodiment 1c, wherein the first purification matrixcomprises (i) a capture domain which binds to the biologic, and (ii) apolypeptide with phase behavior, wherein the capture domain is coupledto the polypeptide with phase behavior.

3c. The method of embodiment 1c, wherein the second purification matrixcomprises (i) a capture domain which binds to the biologic, and (ii) apolypeptide with phase behavior, wherein the capture domain is coupledto the polypeptide with phase behavior.

4c. The method of embodiment 2c or 3c, wherein the capture domain iscoupled to the polypeptide with phase behavior via a linker.

5c. The method of embodiment 4c, wherein the linker is a peptide linker.

6c. The method of embodiment 5c, wherein the peptide linker comprises aprotease cleavage site.

7c. The method of embodiment 4c, wherein the linker is a chemicallinker.

8c. The method of embodiment 1c, wherein the first purification matrixcomprises a fusion protein comprising (i) a capture domain which bindsto the biologic and (ii) a polypeptide with phase behavior.

9c. The method of embodiment 2c, wherein the second purification matrixcomprises a fusion protein comprising (i) a capture domain which bindsto the biologic and (ii) a polypeptide with phase behavior.

10c. The method of any one of embodiments 2c-9c, wherein the polypeptidewith phase behavior is a resilin-like polypeptide.

11c. The method of any one of embodiments 2c-9c, wherein the polypeptidewith phase behavior is an elastin-like polypeptide.

12c. The method of any one of embodiments 2c-9c or 11c, wherein thepolypeptide with phase behavior is a polymer containing a pentapeptiderepeat having the sequence (Val-Pro-Gly-Xaa-Gly)_(n) (SEQ ID NO: 10), ora randomized, scrambled analog thereof; wherein Xaa can be any aminoacid except proline.

13c. The method of embodiment 12c, wherein n is an integer from 1 to360, inclusive of endpoints.

14c. The method of any one of embodiments embodiments 2c-9c or 11c,wherein the polypeptide with phase behavior comprises an amino acidsequence selected from:

-   a. (GRGDSPY)_(n) (SEQ ID NO: 1)-   b. (GRGDSPH)_(n) (SEQ ID NO: 2)-   c. (GRGDSPV)_(n) (SEQ ID NO: 3)-   d. (GRGDSPYG)_(n) (SEQ ID NO: 4)-   e. (RPLGYDS)_(n) (SEQ ID NO: 5)-   f. (RPAGYDS)_(n) (SEQ ID NO: 6)-   g. (GRGDSYP)_(n) (SEQ ID NO: 7)-   h. (GRGDSPYQ)_(n) (SEQ ID NO: 8)-   i. (GRGNSPYG)_(n) (SEQ ID NO: 9)-   j. (GVGVP)_(n) (SEQ ID NO: 11);-   k. (GVGVPGLGVPGVGVPGLGVPGVGVP)_(m) (SEQ ID NO: 12);-   l. (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 13);-   m. (GVGVPGWGVPGVGVPGWGVPGVGVP)_(m) (SEQ ID NO: 14);-   n. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)_(m) (SEQ ID NO:    15);-   o. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)_(m) (SEQ ID NO:    16); and-   p. (GAGVPGVGVPGAGVPGVGVPGAGVP)_(m) (SEQ ID NO: 17);-   or a randomized, scrambled analog thereof;-   wherein:    -   n is an integer in the range of 20-360, inclusive of endpoints;        and    -   m is an integer in the range of 4-25, inclusive of endpoints.

15c. The method of any one of embodiments 2c-9c or 11c, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GVGVP)_(m) (SEQ ID NO: 52);-   b. (ZZPXXXXGZ)_(m) (SEQ ID NO: 57);-   c. (ZZPXGZ)_(m) (SEQ ID NO: 58);-   d. (ZZPXXGZ)_(m) (SEQ ID NO: 59); or-   e. (ZZPXXXGZ)_(m) (SEQ ID NO: 60),-   wherein m is an integer between 10 and 160, inclusive of endpoints,    wherein X if present is any amino acid except proline or glycine,    and wherein Z if present is any amino acid.

16c. The method of any one of embodiments 2c-9c or 11c, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 53); or-   (b) (GVGVPGVGVPGLGVPGVGVPGVGVP)_(m) (SEQ ID NO: 55);-   wherein m is an integer between 2 and 32, inclusive of endpoints.

17c. The method of any one of embodiments 2c-9c or 11c, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 193), wherein m is 8    or 16;-   (b) (GVGVPGAGVP)_(m) (SEQ ID NO: 54), wherein m is an integer    between 5 and 80, inclusive of endpoints; or-   (c) (GXGVP)_(m) (SEQ ID NO: 56), wherein m is an integer between 10    and 160, inclusive of endpoints, and wherein X for each repeat is    independently selected from the group consisting of glycine,    alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,    tryptophan, lysine, arginine, aspartic acid, glutamic acid, and    serine.

18c. The method of any one of embodiments 2c-17c, wherein the capturedomain comprises the sequence of any one of SEQ ID NO: 24-49 and 62-148and 167-171, or a sequence having at least 1, at least 2, at least 3, atleast 4, or at least 5 mutations relative thereto.

19c. The method of any one of embodiments 1c-17c, wherein the binding ofthe first biologic and/or the second biologic to the purification matrixis reversible.

20c. The method of any one of embodiments 1c-17c, wherein the binding ofthe first biologic and/or the second biologic to the purification matrixis non-covalent.

21c. The method of any one of embodiments 1c-17c, wherein the binding ofthe first biologic and/or the second biologic to the purification matrixis covalent.

22c. The method of any one of embodiments 1c-21c, wherein the firstbiologic and/or the second biologic is selected from a cell, a protein,a lipid, a lipopolysaccharide, a nucleic acid, or a viral particle.

23c. The method of embodiment 22c, wherein the first biologic and/or thesecond biologic is a cell.

24c. The method of embodiment 23c, wherein the cell is a bacterial cell,a yeast cell, or a mammalian cell.

25c. The method of embodiment 23c or 24c, wherein the cell is a stemcell, a bone cell, a blood cell, a muscle cell, a fat cell, a skin cell,a nerve cell, an endothelial cell, a sex cell, a pancreatic cell, or acancer cell.

26c. The method of embodiment 23c or 24c, wherein the cell is an immunecell.

27c. The method of embodiment 26c, wherein the immune cell is a T cell,a B cell, a NK cell, a peripheral blood mononuclear cell, or aneutrophil.

28c. The method of embodiment 27c, wherein the cell is a T cellexpressing a chimeric antigen receptor (CAR).

29c. The method of embodiment 22c, wherein the nucleic acid is a DNA oran RNA.

30c. The method of embodiment 22c, wherein the viral particle is anadenovirus particle, an adeno-associated virus (AAV) particle, alentivirus particle, a retrovirus particle, a poxvirus particle, ameasles virus particle or a herpesvirus particle.

31c. The method of any one of embodiments 1c-30c, wherein the firstbiologic and/or second biologic has a diameter between 1 nm and 100 µm,inclusive of the endpoints.

32c. The method of embodiment 31c, wherein the first biologic and/orsecond biologic has a diameter between 1 nm and 100 nm, inclusive of theendpoints.

33c. The method of embodiment 31c, wherein the first biologic and/orsecond biologic has a diameter between 100 nm and 1 µm, inclusive of theendpoints.

34c. The method of embodiment 31c, wherein the first biologic and/orsecond biologic has a diameter between 1 µm and 50 µm, inclusive of theendpoints.

35c. The method of embodiment 31c, wherein the first biologic and/orsecond biologic has a diameter between 50 µm and 100 µm, inclusive ofthe endpoints.

36c. The method of any one of embodiments 1c-35c, wherein the method iscompleted in about 0.5 to about 24 hours.

37c. The method of embodiment 36c, wherein the method is completed inabout 0.5 to about 8 hours.

38c. The method of embodiment 36c, wherein the method is completed inabout 2 to about 6 hours.

39c. The method of any one of embodiments 1c-38c, wherein the separationof the first complex from the second complex can be observed visuallywith an unaided eye.

40c. The method of any one of embodiments 1c-39c, wherein the increasein the size of the first complex and/or second complex is at least a2-fold increase.

41c. The method of any one of embodiments 1c-40c, wherein the increasein the size of the first complex and/or second complex is at least a10-fold increase.

42c. The method of any one of embodiments 1c-41c, wherein the increasein the size of the first complex and/or second complex is at least a25-fold increase.

43c. The method of any one of embodiments 40c-42c, wherein the increasein size is an increase in the mass of the complex.

44c. The method of any one of embodiments 40c-42c, wherein the increasein size is an increase in the diameter of the complex.

45c. The method of any one of embodiments 1c-44c, wherein theenvironmental factor comprises one or more of:

-   a. a change in one or more of temperature, pH, salt concentration,    concentration of the purification matrix, concentration of the    biologic, or pressure;-   b. the addition of one or more surfactants, molecular crowding    agents, reducing agents, oxidizing agents, enzymes, or denaturing    agents; or-   c. the application of electromagnetic or acoustic waves.

46c. The method of any one of embodiments 1c-45c, wherein the separationon the basis of size is performed using tangential flow filtration,analytical ultracentrifugation, membrane chromatography, highperformance liquid chromatography, and fast protein liquidchromatography.

47c. The method of any one of embodiments 1c-46c, wherein thepurification yield of the first biologic is at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%.

48c. The method of any one of embodiments 1c-47c, wherein thepurification yield of the second biologic is at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%.

A Method of Bringing a First Biologic Into Proximity With a SecondBiologic

1d. A method of bringing a first biologic into proximity with a secondbiologic, the method comprising contacting the first biologic with afirst protein-based purification matrix and contacting the secondbiologic with a second protein-based purification matrix;

-   wherein the first biologic binds to the first purification matrix to    form a first complex;-   wherein the second biologic binds to the second purification matrix    to form a second complex; and-   wherein an environmental factor brings the first complex and the    second complex into proximity with one another.

2d. The method of embodiment 1d, wherein the first purification matrixcomprises (i) a capture domain which binds to the biologic, and (ii) apolypeptide with phase behavior, wherein the capture domain is coupledto the polypeptide with phase behavior.

3d. The method of embodiment 1d, wherein the second purification matrixcomprises (i) a capture domain which binds to the biologic, and (ii) apolypeptide with phase behavior, wherein the capture domain is coupledto the polypeptide with phase behavior.

4d. The method of embodiment 2d or 3d, wherein the capture domain iscoupled to the polypeptide with phase behavior via a linker.

5d. The method of embodiment 4d, wherein the linker is a peptide linker.

6d. The method of embodiment 5d, wherein the peptide linker comprises aprotease cleavage site.

7d. The method of embodiment 4d, wherein the linker is a chemicallinker.

8d. The method of embodiment 1d, wherein the first purification matrixcomprises a fusion protein comprising (i) a capture domain which bindsto the biologic and (ii) a polypeptide with phase behavior.

9d. The method of embodiment 1d, wherein the second purification matrixcomprises a fusion protein comprising (i) a capture domain which bindsto the biologic and (ii) a polypeptide with phase behavior.

10d. The method of any one of embodiments 1d-9d, wherein the polypeptidewith phase behavior is a resilin-like polypeptide.

11d. The method of any one of embodiments 1d-9d, wherein the polypeptidewith phase behavior is an elastin-like polypeptide.

12d. The method of any one of embodiments 1d-9d or 11d, wherein thepolypeptide with phase behavior is a polymer containing a pentapeptiderepeat having the sequence (Val-Pro-Gly-Xaa-Gly)_(n) (SEQ ID NO: 10), ora randomized, scrambled analog thereof; wherein Xaa can be any aminoacid except proline.

13d. The method of embodiment 12d, wherein n is an integer from 1 to360, inclusive of endpoints.

14d. The method of any one of embodiments 1d-9d or 11d, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GRGDSPY)_(n) (SEQ ID NO: 1)-   b. (GRGDSPH)_(n) (SEQ ID NO: 2)-   c. (GRGDSPV)_(n) (SEQ ID NO: 3)-   d. (GRGDSPYG)_(n) (SEQ ID NO: 4)-   e. (RPLGYDS)_(n) (SEQ ID NO: 5)-   f. (RPAGYDS)_(n) (SEQ ID NO: 6)-   g. (GRGDSYP)_(n) (SEQ ID NO: 7)-   h. (GRGDSPYQ)_(n) (SEQ ID NO: 8)-   i. (GRGNSPYG)_(n) (SEQ ID NO: 9)-   j. (GVGVP)_(n) (SEQ ID NO: 11);-   k. (GVGVPGLGVPGVGVPGLGVPGVGVP)_(m) (SEQ ID NO: 12);-   l. (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 13);-   m. (GVGVPGWGVPGVGVPGWGVPGVGVP)_(m) (SEQ ID NO: 14);-   n. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)_(m) (SEQ ID NO:    15);-   o. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)_(m) (SEQ ID NO:    16); and-   p. (GAGVPGVGVPGAGVPGVGVPGAGVP)_(m) (SEQ ID NO: 17);-   or a randomized, scrambled analog thereof;-   wherein:    -   n is an integer in the range of 20-360, inclusive of endpoints;        and    -   m is an integer in the range of 4-25, inclusive of endpoints.

15d. The method of any one of embodiments 1d-9d or 11d, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   a. (GVGVP)_(m) (SEQ ID NO: 52);-   b. (ZZPXXXXGZ)_(m) (SEQ ID NO: 57);-   c. (ZZPXGZ)_(m)(SEQ ID NO: 58);-   d. (ZZPXXGZ)_(m) (SEQ ID NO: 59); or-   e. (ZZPXXXGZ)_(m) (SEQ ID NO: 60),-   wherein m is an integer between 10 and 160, inclusive of endpoints,    wherein X if present is any amino acid except proline or glycine,    and wherein Z if present is any amino acid.

16d. The method of any one of embodiments 1d-9d or 11d, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 53); or-   (b) (GVGVPGVGVPGLGVPGVGVPGVGVP)_(m) (SEQ ID NO: 55);-   wherein m is an integer between 2 and 32, inclusive of endpoints.

17d. The method of any one of embodiments 1d-9d or 11d, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from:

-   (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)_(m) (SEQ ID NO: 193), wherein m is 8    or 16;-   (b) (GVGVPGAGVP)_(m) (SEQ ID NO: 54), wherein m is an integer    between 5 and 80, inclusive of endpoints; or-   (c) (GXGVP)_(m) (SEQ ID NO: 56), wherein m is an integer between 10    and 160, inclusive of endpoints, and wherein X for each repeat is    independently selected from the group consisting of glycine,    alanine, valine, isoleucine, leucine, phenylalanine, tyrosine,    tryptophan, lysine, arginine, aspartic acid, glutamic acid, and    serine.

18d. The method of any one of embodiments 2d-17d, wherein the capturedomain comprises the sequence of any one of SEQ ID NO: 24-49 and 62-148and 167-171, or a sequence having at least 1, at least 2, at least 3, atleast 4, or at least 5 mutations relative thereto.

19d. The method of any one of embodiments 1d-18d, wherein the binding ofthe first biologic and/or second biologic to the purification matrix isreversible.

20d. The method of any one of embodiments 1d-18d, wherein the binding ofthe first biologic and/or second biologic to the purification matrix isnon-covalent.

21d. The method of any one of embodiments 1d-18d, wherein the binding ofthe first biologic and/or second biologic to the purification matrix iscovalent.

22d. The method of any one of embodiments 1d-21d, wherein the firstbiologic and/or second biologic is selected from a cell, a protein, alipid, a lipopolysaccharide, a nucleic acid, a carbohydrate, or a viralparticle.

23d. The method of embodiment 22d, wherein the first biologic and/orsecond biologic is a cell.

24d. The method of embodiment 23d, wherein the cell is a bacterial cell,a yeast cell, or a mammalian cell.

25d. The method of embodiment 23d or 24d, wherein the cell is a stemcell, a bone cell, a blood cell, a muscle cell, a fat cell, a skin cell,a nerve cell, an endothelial cell, a sex cell, a pancreatic cell, or acancer cell.

26d. The method of any one of embodiments 23d or 24d, wherein the cellis an immune cell.

27d. The method of embodiment 26d, wherein the immune cell is a T cell,a B cell, a NK cell, a peripheral blood mononuclear cell, or aneutrophil.

28d. The method of embodiment 27d, wherein the cell is a T cellexpressing a chimeric antigen receptor (CAR).

29d. The method of embodiment 22d, wherein the nucleic acid is a DNA oran RNA.

30d. The method of embodiment 22d, wherein the viral particle is anadenovirus particle, an adeno-associated virus (AAV) particle, alentivirus particle, a retrovirus particle, a poxvirus particle, ameasles virus particle, or a herpesvirus particle.

31d. The method of any one of embodiments 1d-30d, wherein the firstbiologic and/or second biologic has a diameter between 1 nm and 100 µm,inclusive of the endpoints.

32d. The method of embodiment 31d, wherein the first biologic and/orsecond biologic has a diameter between 1 nm and 100 nm, inclusive of theendpoints.

33d. The method of embodiment 31d, wherein the first biologic and/orsecond biologic has a diameter between 100 nm and 1 µm, inclusive of theendpoints.

34d. The method of embodiment 31d, wherein the first biologic and/orsecond biologic has a diameter between 1 µm and 50 µm, inclusive of theendpoints.

35d. The method of embodiment 31d, wherein the first biologic and/orsecond biologic has a diameter between 50 µm and 100 µm, inclusive ofthe endpoints.

36d. The method of any one of embodiments 1d-35d, wherein the method iscompleted in about 0.5 to about 24 hours.

37d. The method of embodiment 36d, wherein the method is completed inabout 0.5 to about 8 hours.

38d. The method of embodiment 36d, wherein the method is completed inabout 2 to about 6 hours.

39d. The method of any one of embodiments 1d-38d, wherein the bringingtogether of the first complex and the second complex can be observedvisually with an unaided eye.

40d. The method of any one of embodiments 1d-39d, wherein the increasein the size of the first complex and/or second complex is at least a2-fold increase.

41d. The method of any embodiment 40d, wherein the increase in the sizeof the first complex and/or second complex is at least a 10-foldincrease.

42d. The method of embodiment 41d, wherein the increase in the size ofthe first complex and/or second complex is at least a 25-fold increase.

43d. The method of any one of embodiments 40d-42d, wherein the increasein size is an increase in the mass of the complex.

44d. The method of any one of embodiments 40d-42d, wherein the increasein size is an increase in the diameter of the complex.

45d. The method of any one of embodiments 1d-44d, wherein theenvironmental factor comprises one or more of:

-   a. a change in one or more of temperature, pH, salt concentration,    concentration of the purification matrix, concentration of the    biologic, or pressure;-   b. the addition of one or more surfactants, molecular crowding    agents, reducing agents, oxidizing agents, cofactor, vitamin,    enzymes, or denaturing agents; or-   c. the application of electromagnetic or acoustic waves.

46d. The method of any one of embodiments 1d-45d, wherein the separationon the basis of size is performed using tangential flow filtration,membrane chromatography, analytical ultracentrifugation, highperformance liquid chromatography, normal flow filtration, acoustic waveseparation, centrifugation, counterflow centrifugation, and fast proteinliquid chromatography.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as, an acknowledgment orany form of suggestion that it constitutes valid prior art or form partof the common general knowledge in any country in the world.

What is claimed is: 1-28. (canceled)
 29. A method of purifying a viralparticle comprising: (i) contacting the viral particle with a fusionprotein comprising (a) at least one low-density lipoprotein receptor(LDLR) or an extracellular domain thereof; and (b) at least onepolypeptide with phase behavior; wherein the fusion protein binds to theviral particle and forms a complex (ii) contacting the complex with afirst environmental factor; (iii) separating the complex from at leastone contaminant; and (iv) separating the viral particle from the fusionprotein by contacting the complex with a second environmental factor.30. The method of claim 29, wherein the LDLR or an extracellular domainthereof comprises the sequence of any one of SEQ ID NO: 73-76 or168-169, or a sequence with at least 90% identity thereto.
 31. Themethod of claim 29, wherein the LDLR or an extracellular domain thereofcomprises the sequence of any one of SEQ ID NO: 73-76 or 168-169, or asequence with at least 95% identity thereto.
 32. The method of claim 29,wherein the LDLR or an extracellular domain thereof comprises thesequence of any one of SEQ ID NO: 73-76 or 168-169, or a sequence withat least 97% identity thereto.
 33. The method of claim 29, wherein theLDLR or an extracellular domain thereof comprises the sequence of anyone of SEQ ID NO: 73-76 or 168-169, or a sequence with at least 99%identity thereto.
 34. The method of claim 29, wherein the LDLR or anextracellular domain thereof comprises the amino acid sequence of anyone of: (a) amino acids 2-860 of SEQ ID NO: 73; (b) amino acids 2-768 ofSEQ ID NO: 74; (c) amino acids 2-40 of SEQ ID NO: 75; (d) amino acids2-38 of SEQ ID NO: 76; (e) SEQ ID NO: 168; or (f) SEQ ID NO:
 169. 35.The method of claim 29, wherein the LDLR or an extracellular domainthereof comprises the amino acid sequence of SEQ ID NO:
 169. 36. Themethod of claim 29, wherein the polypeptide with phase behavior is anelastin-like polypeptide (ELP).
 37. The method of claim 29, wherein theELP is a polymer containing a pentapeptide repeat having the sequence(Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 10); wherein Xaa can be any aminoacid except proline; and wherein n is an integer from 1 to 360,inclusive of endpoints.
 38. The method of claim 29, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from: (a) (GRGDSPY)n (SEQ ID NO: 1); (b) (GRGDSPH)n (SEQ ID NO:2); (c) (GRGDSPV)n (SEQ ID NO: 3); (d) (GRGDSPYG)n (SEQ ID NO: 4); (e)(RPLGYDS)n (SEQ ID NO: 5); (f) (RPAGYDS)n (SEQ ID NO: 6); (g) (GRGDSYP)n(SEQ ID NO: 7); (h) (GRGDSPYQ)n (SEQ ID NO: 8); (i) (GRGNSPYG)n (SEQ IDNO: 9); (j) (GVGVP)n (SEQ ID NO: 11); (k) (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12); (1) (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 13); (m)(GVGVPGWGVPGVGVPGWGVPGVGVP)m (SEQ ID NO: 14); (n)(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m (SEQ ID NO: 15); (o)(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m (SEQ ID NO: 16); and(p) (GAGVPGVGVPGAGVPGVGVPGAGVP)m (SEQ ID NO: 17); wherein n is aninteger in the range of 20-360, inclusive of endpoints; and m is aninteger in the range of 4-25, inclusive of endpoints.
 39. The method ofclaim 29, wherein the polypeptide with phase behavior comprises an aminoacid sequence selected from: (a) (GVGVP)m (SEQ ID NO: 52); (b)(ZZPXXXXGZ)m (SEQ ID NO: 57); (c) (ZZPXGZ)m (SEQ ID NO: 58); (d)(ZZPXXGZ)m (SEQ ID NO: 59); or (e) (ZZPXXXGZ)m (SEQ ID NO: 60); whereinm is an integer between 10 and 160, inclusive of endpoints; wherein X ifpresent is any amino acid except proline or glycine; and wherein Z ifpresent is any amino acid.
 40. The method of claim 29, wherein thepolypeptide with phase behavior comprises an amino acid sequenceselected from: (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 53); and (b)(GVGVPGVGVPGLGVPGVGVPGVGVP)m (SEQ ID NO: 55); wherein m is an integerbetween 2 and 32, inclusive of endpoints.
 41. The method of claim 29,wherein the polypeptide with phase behavior comprises an amino acidsequence selected from any one of: (a) (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQID NO: 193), wherein m is 8 or 16; (b) (GVGVPGAGVP)m (SEQ ID NO: 54),wherein m is an integer between 5 and 80, inclusive of endpoints; and(c) (GXGVP)m (SEQ ID NO: 56), wherein m is an integer between 10 and160, inclusive of endpoints, wherein each X is independently selectedfrom the group consisting of glycine, alanine, valine, isoleucine,leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, asparticacid, glutamic acid, and serine.
 42. The method of claim 29, wherein thepolypeptide with phase behavior comprises the amino acid sequence of(GVGVP)m, wherein m is
 80. 43. The method of claim 35, wherein: (a) thepolypeptide with phase behavior comprises the amino acid sequence of(GVGVP)m, wherein m is 80; and (b) wherein the LDLR or an extracellulardomain thereof comprises the amino acid sequence of SEQ ID NO:
 169. 44.The method of claim 29, comprising adding the fusion protein to tissueculture media of cells producing the viral particle.
 45. The method ofclaim 29, wherein the first environmental factor is selected from thegroup consisting of: a change in temperature, a change in pH, a changein salt concentration, a change in the concentration of the fusionprotein, a change in the concentration of the viral particle, a changein pressure, the addition of a surfactant, the addition of a cofactor,the addition of a vitamin, the addition of a molecular crowding agent,the addition of a reducing agent, the addition of an oxidizing agent,the addition of an enzyme, the addition of a denaturing agent, theapplication of electromagnetic waves, the application of acoustic waves,or a combination thereof.
 46. The method of claim 29, wherein the secondenvironmental factor is selected from the group consisting of: a changein temperature, a change in pH, a change in salt concentration, a changein the concentration of the fusion protein, a change in theconcentration of the viral particle, a change in pressure, the additionof a surfactant, the addition of a cofactor, the addition of a vitamin,the addition of a molecular crowding agent, the addition of a reducingagent, the addition of an oxidizing agent, the addition of an enzyme,the addition of a denaturing agent, the application of electromagneticwaves, the application of acoustic waves, or a combination thereof. 47.The method of claim 29, comprising separating the complex from at leastone contaminant by a method selected from the group consisting of:tangential flow filtration, membrane chromatography, analyticalultracentrifugation, high performance liquid chromatography, membranechromatography, normal flow filtration, acoustic wave separation,centrifugation, counterflow centrifugation, and fast protein liquidchromatography.
 48. The method of claim 29, wherein the firstenvironmental factor is the addition of salt.
 49. The method of claim29, wherein the purification yield of the viral particle is at least 70%.